Immunogenic composition

ABSTRACT

The present application relates to immunogenic compositions comprising staphylococcal PNAG and Type 5 and/or 8 capular polysaccharide or oligosaccharide from  S. aureus . Vaccines, methods of treatment using and processes to make an immunogenic composition comprising PNAG and Type 5 and/or 8 capsular polysaccharides are also described.

TECHNICAL FIELD

The present invention relates to the field of Staphylococcal immunogeniccompositions and vaccines, their manufacture and the use of suchcompositions in medicine. More particularly, it relates to vaccinecompositions comprising PNAG (PIA) polysaccharide and type 5 and/or 8polysaccharides from S. aureus. Methods for the treatment or preventionof staphylococcal infections using such vaccines are also provided.

BACKGROUND

The number of both community acquired and hospital acquired infectionshave increased over recent years with the increased use of intravasculardevices. Hospital acquired (nosocomial) infections are a major cause ofmorbidity and mortality, more particularly in the US, where if affectsmore than 2 million patients annually. Following various studies, about6 percent of the US patients will acquire an infection during their stayin hospital. The economic burden in the USA was estimated to be morethan $4.5 billion in 1992 (Emori and Gaynes, 1993, Clin. Microbiol. Rev.6; 428). The most frequent infections are urinary tract infections(UTI-33% of the infections), followed by pneumonia (15.5%), surgicalsite infections (14.8%) and primary bloodstream infections (13%) Emoriand Gaynes, 1993, Clin. Microbiol. Rev. 6; 428).

Staphylococcus aureus, Coagulase-negative Staphylococci (mostlyStaphylococcus epidermidis), enterococcus spp, Esherichia coli andPseudomonas aeruginosa are the major nosocomial pathogens. Althoughthose pathogens almost cause the same number of infections, the severityof the disorders they can produce combined with the frequency ofantibiotic resistant isolates balance this ranking towards S. aureus andS. epidermidis as being the most significant nosocomial pathogens.

Staphylococcus aureus is the most common cause of nosocomial infectionswith a significant morbidity and mortality (Romero-Vivas et al 1995,Infect. Dis. 21; 1417). It is the cause of some cases of osteomyelitis,endocarditis, septic arthritis, pneumonia, abscesses and toxic shocksyndrome.

S. epidermidis is a normal skin commensal which is also an importantopportunistic pathogen responsible for infections of implanted medicaldevices and infections at sites of surgery. Medical devices infected byS. epidermidis include cardiac pacemakers, cerebrospinal fluid shunts,continuous ambulatory peritoneal dialysis catheters, orthopaedic devicesand prosthetic heart valves.

S. aureus and S. epidermidis infections are treated with antibiotics,with penicillin being the drug of choice whereas vancomycin is used formethicillin resistant isolates. The percentage of staphylococcal strainsexhibiting wide-spectrum resistance to antibiotics has becomeincreasingly prevalent since the 1980's (Panlilo et al 1992, Infect.Control. Hosp. Epidemiol. 13; 582), posing a threat for effectiveantimicrobial therapy. In addition, the recent emergence of vancomycinresistant S. aureus strain has aroused fear that methicillin resistantS. aureus strains will emerge and spread for which no effective therapyis available.

An alternative approach of using antibodies against staphylococcalantigens in passive immunotherapy has been investigated. Therapyinvolving administration of polyclonal antisera are under development(WO 00/15238, WO 00/12132) as well as treatment with a monoclonalantibody against lipoteichoic acid (WO 98/57994).

An alternative approach would be use of active vaccination to generatean immune response against staphylococci. Several candidates forinclusion as vaccine components have been identified. These includeFibronectin binding protein (U.S. Pat. No. 5,840,846), MHC II analogue(U.S. Pat. No. 5,648,240), fibrinogen binding protein (U.S. Pat. No.6,008,341), GehD (US 2002/0169288), collagen binding protein (U.S. Pat.No. 6,288,214), SdrF, SdrG and SdrH (WO 00/12689), mutant SEA and SEBexotoxins (WO 00/02523) and 52 kDa vitronectin binding protein (WO01/60852).

The S. aureus genome has been sequenced and many of the coding sequenceshave been identified (EP786519, WO02/094868). The same is true for S.epidermidis (WO 01/34809). As a refinement of this approach, others haveidentified proteins that are recognised by hyperimmune sera frompatients who have suffered staphylococcal infection (WO01/98499, WO02/059148).

The first generation of vaccines targeted against S. aureus or againstthe exoproteins it produces have met with limited success (Lee 1996Trends Microbiol. 4; 162). There remains a need to develop effectivevaccines against staphylococcal infections.

DESCRIPTION OF FIGURES

FIG. 1—Polypeptide sequences of preferred proteins. Table 1 providesinformation on which protein is represented by each SEQ ID.

FIG. 2—Nucleotide sequences encoding preferred proteins. Table 1provides information on which protein is encoded by each SEQ ID.

FIG. 3—Purification of alpha toxin under native conditions. Panel Ashows a coommassie stained SDS-PAGE of samples prepared during thepurification of alpha toxin. Lane 1—molecular weight markers, lane2—soluble fraction containing over-expressed alpha toxin, lane 3—flowthrough from the Ni-NTA column, lane 4—fractions eluted with 10% bufferB, lane 5—fractions eluted with 20% buffer B, lane 6—fractions elutedwith 30% buffer B, lane 7—fractions eluted with 50% buffer B, lane8—fractions eluted with 75% buffer B, lane 9 and 10 fractions elutedwith 100% buffer B, lane 11 bacteria at T=0 before induction, lane12—bacteria at T=4 hours after induction, lane 13—cell lysate, lane14—soluble fraction, lane 15—insoluble fraction.

Panel B shows a coommassie stained SDS-PAGE of 10, 5, 2 and 1 μl of thepurified alpha toxin.

FIG. 4—Purification of SdrC underdenaturing conditions. Panel A shows acoommassie stained SDS-PAGE of samples prepared during the purificationof alpha toxin. Lane M—molecular weight markers, lane Start—supernatantformed from the insoluble fraction containing over-expressed SdrC, laneFT1—flow through from the Ni-NTA column, lane C—fractions eluted withwash buffer C, lane D—fractions eluted with buffer D, lane E—fractionseluted with buffer E.

Panel B shows a coommassie stained SDS-PAGE of 1, 2, 5 and 10 μl of thepurified SdrC.

FIG. 5—ELISA results for antisera against staphylococcal proteins inplates coated with purified proteins.

Pool mice pre—result using pooled sera extracted from micepre-innoculation. Pool mice Post III—result using pooled mouse seraextracted post-immunisation. Pool rabbit pre—result using pooled seraextracted from rabbits pre-innoculation. Pool rabbit Post III—resultusing pooled rabbit sera extracted post-immunisation. BIc—negativecontrol.

FIG. 6—ELISA results for mouse antisera raised against staphylococcalproteins in plates coated with killed staphylococci.

Panel A uses plates coated with S. aureus serotype 5 killed whole cells.Panel B uses plates coated with S. aureus serotype 8 killed whole cells.Panel C uses plates coated with S. epidermidis killed whole cells.

The line marked with square signs shows the ELISA result using antiserafrom mice immunised three times with the indicated staphylococcalprotein. The line marked with diamond signs shows the ELISA result forpre-immune mouse sera.

FIG. 7—ELISA results for rabbit antisera raised against staphylococcalproteins in plates coated with killed staphylococci.

Panel A uses plates coated with S. aureus serotype 5 killed whole cells.Panel B uses plates coated with S. aureus serotype 8 killed whole cells.Panel C uses plates coated with S. epidermidis killed whole cells.

The line marked with square signs shows the ELISA result using antiserafrom rabbits immunised three times with the indicated staphylococcalprotein (except for HarA where only one immunisation was given). Theline marked with diamond signs shows the ELISA result for pre-immunerabbit sera.

DETAILED DESCRIPTION

The present invention discloses particular combinations ofStaphylococcal antigens which when combined, lead to the production ofan immunogenic composition for treating or preventing staphylococcalinfection. Immunogenic compositions of the invention incorporate PNAG(PIA) and S. aureus polysaccharides type 5 and/or 8. This combination ofantigens is capable of eliciting an immune response against a range ofstaphylococcal infections. PNAG (PIA) is highly conserved among Grampositive bacteria and provides protection against a broad range ofbacteria whereas Type 5 and 8 polysaccharides are potent immunogens thatelicit an immune response against most strains of S. aureus which is themost common cause of nosocomial infection.

Polysaccharides

The immunogenic compositions of the invention comprise PIA (also knownas PNAG) and type 5 and 8 polysaccharides from S. aureus.

PIA (PNAG)

It is now clear that the various forms of staphylococcal surfacepolysaccharides identified as PS/A, PIA and SAA are the same chemicalentity—PNAG (Maira-Litran et al Vaccine 22; 872-879 (2004)). Thereforethe term PIA or PNAG encompasses all these polysaccharides oroligosaccharides derived from them.

PIA is a polysaccharide intercellular adhesin and is composed of apolymer of β-(1→6)-linked glucosamine substituted with N-acetyl andO-succinyl constituents. This polysaccharide is present in both S.aureus and S. epidermidis and can be isolated from either source (Joyceet al 2003, Carbohydrate Research 338; 903; Maira-Litran et al 2002,Infect. Imun. 70; 4433). For example, PNAG may be isolated from S.aureus strain MN8m (WO 04/43407).

PIA isolated from S. epidermidis is a integral constituent of biofilm.It is responsible for mediating cell-cell adhesion and probably alsofunctions to shield the growing colony from the host's immune response.

The polysaccharide previously known aspoly-N-succinyl-β-(1→6)-glucosamine (PNSG) was recently shown not tohave the expected structure since the identification of N-succinylationwas incorrect (Maira-Litran et al 2002, Infect. Imun. 70; 4433).Therefore the polysaccharide formally known as PNSG and now found to bePNAG is also encompassed by the term PIA.

PIA (or PNAG) may be of different sizes varying from over 400 kDa tobetween 75 and 400 kDa to between 10 and 75 kDa to oligosaccharidescomposed of up to 30 repeat units (of β-(1→6)-linked glucosaminesubstituted with N-acetyl and O-succinyl constituents). Any size of PIApolysaccharide or oligosaccharide may be use in an immunogeniccomposition of the invention, however a size of over 40 kDa ispreferred. Sizing may be achieved by any method known in the art, forinstance by microfluidisation, ultrasonic irradiation or by chemicalcleavage (WO 03/53462, EP497524, EP497525).

Preferred size ranges of PIA (PNAG) are 40-400 kDa, 50-350 kDa, 40-300kDa, 60-300 kDa, 50-250 kDa and 60-200 kDa.

PIA (PNAG) can have different degree of acetylation due to substitutionon the amino groups by acetate. PIA produced in vitro is almost fullysubstituted on amino groups (95-100%). Alternatively, a deacetylated PIA(PNAG) can be used having less than 60%, preferably less than 50%, 40%,30%, 20%, 10% acetylation. Use of a deacetylated PIA (PNAG) is preferredsince non-acetylated epitopes of PNAG are efficient at mediating opsonickilling of Gram positive bacteria, preferably S. aureus and/or S.epidermidis. Most preferably, the PIA (PNAG) has a size between 40 kDaand 300 kDa and is deacetylated so that less than 60%, 50%, 40%, 30% or20% of amino groups are acetylated.

The term deacetylated PNAG (dPNAG) refers to a PNAG polysaccharide oroligosaccharide in which less than 60%, 50%, 40%, 30%, 20% or 10% of theamino agroups are acetylated.

In an embodiment, PNAG is a deaceylated to form dPNAG by chemicallytreating the native polysaccharide. For example, the native PNAG istreated with a basic solution such that the pH rises to above 10. Forinstance the PNAG is treated with 0.1-5M, 0.2-4M, 0.3-3M, 0.5-2M,0.75-1.5M or 1M NaOH, KOH or NH₄OH. Treatment is for at least 10 or 30minutes, or 1, 2, 3, 4, 5, 10, 15 or 20 hours at a temperature of20-100, 25-80, 30-60 or 30-50 or 35-45° C. dPNAG may be prepared asdescribed in WO 04/43405.

The polysaccharide(s) included in the immunogenic composition of theinvention are preferably conjugated to a carrier protein as describedbelow or alternatively unconjugated.

Type 5 and Type 8 Polysaccharides from S. aureus

Most strains of S. aureus that cause infection in man contain eitherType 5 or Type 8 polysaccharides. Approximately 60% of human strains areType 8 and approximately 30% are Type 5. The structures of Type 5 andType 8 capsular polysaccharide antigens are described in Moreau et alCarbohydrate Res. 201; 285 (1990) and Fournier et al Infect. Immun. 45;87 (1984). Both have FucNAcp in their repeat unit as well as ManNAcAwhich can be used to introduce a sulfhydryl group. The structures werereported as:

Type 5 →4)-β-D-ManNAcA(3OAc)-(1→4)-α-L-FucNAc(1→3)-β-D-FucNAc-(1→ Type 8→3)-β-D-ManNAcA(4OAc)-(1→3)-α-L-FucNAc(1→3)-β-D-FucNAc-(1→

Recently (Jones Carbohydrate Research 340, 1097-1106 (2005)) NMRspectroscopy revised to structures to:

Type 5 →4)-β-D-ManNAcA-(1→4)-α-L-FucNAc(3OAc)-(1→3)-β-D-FucNAc-(1→ Type8 →3)-β-D-ManNAcA(4OAc)-(1→3)-α-L-FucNAc(1→3)-α-D-FucNAc(1→

Polysaccharides may be extracted from the appropriate strain of S.aureus using method well known to the skilled man, for instance asdescribed in U.S. Pat. No. 6,294,177. For example, ATCC 12902 is a Type5 S. aureus strain and ATCC 12605 is a Type 8 S. aureus strain.

Polysaccharides are of native size or alternatively may be sized, forinstance by microfluidisation, ultrasonic irradiation or by chemicaltreatment. The invention also covers oligosaccharides derived from thetype 5 and 8 polysaccharides from S. aureus.

The type 5 and 8 polysaccharides included in the immunogenic compositionof the invention are preferably conjugated to a carrier protein asdescribed below or are alternatively unconjugated.

The immunogenic compositions of the invention alternatively containseither type 5 or type 8 polysaccharide.

S. aureus 336 Antigen

In an embodiment, the immunogenic composition of the invention comprisesthe S. aureus 336 antigen described in U.S. Pat. No. 6,294,177.

The 336 antigen comprises β-linked hexosamine, contains no O-acetylgroups and specifically binds to antibodies to S. aureus Type 336deposited under ATCC 55804.

In an embodiment, the 336 antigen is a polysaccharide which is of nativesize or alternatively may be sized, for instance by microfluidisation,ultrasonic irradiation or by chemical treatment. The invention alsocovers oligosaccharides derived from the 336 antigen.

The 336 antigen, where included in the immunogenic composition of theinvention is preferably conjugated to a carrier protein as describedbelow or are alternatively unconjugated.

Type I, II and III Polysaccharides from S. epidermidis

Strains ATCC-31432, SE-360 and SE-10 of S. epidermidis arecharacteristic of three different capsular types, I, II and IIIrespectively (Ichiman and Yoshida 1981, J. Appl. Bacteriol. 51; 229).Capsular polysaccharides extracted from each serotype of S. epidermidisconstitute Type I, II and III polysaccharides. Polysaccharides may beextracted by several methods including the method described in U.S. Pat.No. 4,197,290 or as described in Ichiman et al 1991, J. Appl. Bacteriol.71; 176.

In one embodiment of the invention, the immunogenic compositioncomprises type I and/or II and/or III polysaccharides oroligosaccharides from S. epidermidis.

Polysaccharides are of native size or alternatively may be sized, forinstance by microfluidisation, ultrasonic irradiation or chemicalcleavage. The invention also covers oligosaccharides extracted from S.epidermidis strains.

These polysaccharides are unconjugated or are preferably conjugated asdescribed below.

Conjugation of Polysaccharides

Amongst the problems associated with the use of polysaccharides invaccination, is the fact that polysaccharides per se are poorimmunogens. Strategies, which have been designed to overcome this lackof immunogenicity, include the linking of the polysaccharide to largeprotein carriers, which provide bystander T-cell help. It is preferredthat the polysaccharides utilised in the invention are linked to aprotein carrier which provide bystander T-cell help. Examples of thesecarriers which are currently used for coupling to polysaccharide oroligosaccharide immunogens include the Diphtheria and Tetanus toxoids(DT, DT Crm197 and TT), Keyhole Limpet Haemocyanin (KLH), Pseudomonasaeruginosa exoprotein A (rEPA) and the purified protein derivative ofTuberculin (PPD), protein D from Haemophilus influenzae, pneumolysin orfragments of any of the above. Fragments suitable for use includefragments encompassing T-helper epitopes. In particular protein Dfragment will preferably contain the N-terminal ⅓ of the protein.Protein D is an IgD-binding protein from Haemophilus influenzae (EP 0594 610 B1).

Despite the common use of these carriers and their success in theinduction of anti polysaccharide antibody responses they are associatedwith several drawbacks. For example, it is known that antigen specificimmune responses may be suppressed by the presence of pre-existingantibodies directed against the carrier, in this case Tetanus toxin (DiJohn et al; Lancet, Dec. 16, 1989). In the population at large, a veryhigh percentage of people will have pre-existing immunity to both DT andTT as people are routinely vaccinated with these antigens. In the UK forexample 95% of children receive the DTP vaccine comprising both DT andTT. Other authors have described the problem of epitope suppression topeptide vaccines in animal models (Sad et al, Immunology, 1991;74:223-227; Schutze et al, J. Immunol. 135: 4, 1985; 2319-2322).

KLH is known as potent immunogen and has already been used as a carrierfor IgE peptides in human clinical trials. However, some adversereactions (DTH-like reactions or IgE sensitisation) as well as antibodyresponses against antibody have been observed.

An alternative carrier protein to use in the immunogenic composition ofthe invention is a single staphylococcal protein or fragment thereof ora fusion protein comprising at least or exactly 1, 2, 3 or 4 or more ofthe staphylococcal proteins listed in the section below or fragmentsthereof.

A new carrier protein that would be particularly advantageous to use inthe context of a staphylococcal vaccine is staphylococcal alpha toxoid.The native form may be conjugated to a polysaccharide since the processof conjugation reduces toxicity.

Preferably a genetically detoxified alpha toxin such as the His35Leu orH is 35 Arg variants are used as carriers since residual toxicity islower. Alternatively the alpha toxin is chemically detoxified bytreatment with a cross-linking reagent, formaldehyde or glutaraldehyde.A genetically detoxified alpha toxin is optionally chemicallydetoxified, preferably by treatment with a cross-linking reagent,formaldehyde or glutaraldehyde to further reduce toxicity. Otherstaphylococcal proteins or fragments thereof, particularly those listedabove may be used as a carrier protein for the polysaccharides listedabove. The carrier protein may be a fusion protein comprising at leastor exactly 1, 2, 3, 4 or 5 of the staphylococcal proteins listed above.

The polysaccharides may be linked to the carrier protein(s) by any knownmethod (for example, by Likhite, U.S. Pat. No. 4,372,945 by Armor etal., U.S. Pat. No. 4,474,757, and Jennings et al., U.S. Pat. No.4,356,170). Preferably, CDAP conjugation chemistry is carried out (seeWO95/08348).

In CDAP, the cyanylating reagent 1-cyano-dimethylaminopyridiniumtetrafluoroborate (CDAP) is preferably used for the synthesis ofpolysaccharide-protein conjugates. The cyanilation reaction can beperformed under relatively mild conditions, which avoids hydrolysis ofthe alkaline sensitive polysaccharides. This synthesis allows directcoupling to a carrier protein.

The polysaccharide may be solubilized in water or a saline solution.CDAP may be dissolved in acetonitrile and added immediately to thepolysaccharide solution. The CDAP reacts with the hydroxyl groups of thepolysaccharide to form a cyanate ester. After the activation step, thecarrier protein is added. Amino groups of lysine react with theactivated polysaccharide to form an isourea covalent link. After thecoupling reaction, a large excess of glycine is then added to quenchresidual activated functional groups. The product is then passed througha gel permeation column to remove unreacted carrier protein and residualreagents.

Proteins

The immunogenic composition of the invention preferably furthercomprises a staphylococcal protein, more preferably a protein from S.aureus or S. epidermidis. Some embodiments of the invention containproteins from both S. aureus and S. epidermidis. Immunogeniccompositions of the invention comprise an isolated protein whichcomprises an amino acid sequence which has at least 85% identity,preferably at least 90% identity, more preferably at least 95% identity,most preferably at least 97-99% or exact identity, to that of anysequence of FIG. 1.

Where a protein is specifically mentioned herein, it is preferably areference to a native or recombinant, full-length protein or optionallya mature protein in which any signal sequence has been removed. Theprotein may be isolated directly from the staphylococcal strain orproduced by recombinant DNA techniques. Immunogenic fragments of theprotein may be incorporated into the immunogenic composition of theinvention. These are fragments comprising at least 10 amino acids,preferably 20 amino acids, more preferably 30 amino acids, morepreferably 40 amino acids or 50 amino acids, most preferably 100 aminoacids, taken contiguously from the amino acid sequence of the protein.In addition, such immunogenic fragments are typically immunologicallyreactive with antibodies generated against the Staphylococcal proteinsor with antibodies generated by infection of a mammalian host withStaphylococci or contain T cell epitopes. Immunogenic fragments alsoincludes fragments that when administered at an effective dose, (eitheralone or as a hapten bound to a carrier), elicit a protective immuneresponse against Staphylococcal infection, more preferably it isprotective against S. aureus and/or S. epidermidis infection. Such animmunogenic fragment may include, for example, the protein lacking anN-terminal leader sequence, and/or a transmembrane domain and/or aC-terminal anchor domain. In a preferred aspect the immunogenic fragmentaccording to the invention comprises substantially all of theextracellular domain of a protein which has at least 85% identity,preferably at least 90% identity, more preferably at least 95% identity,most preferably at least 97-99% identity, to that a sequence selectedfrom FIG. 1 over the entire length of the fragment sequence.

In an embodiment, immunogenic compositions of the invention may containfusion proteins of Staphylococcal proteins, or fragments ofstaphylococcal proteins. Such fusion proteins may be made recombinantlyand may comprise one portion of at least 2, 3, 4, 5 or 6 staphylococcalproteins. Alternatively, a fusion protein may comprise multiple portionsof at least 2, 3, 4 or 5 staphylococcal proteins. These may combinedifferent Staphylococcal proteins or fragments thereof in the sameprotein. Alternatively, the invention also includes individual fusionproteins of Staphylococcal proteins or fragments thereof, as a fusionprotein with heterologous sequences such as a provider of T-cellepitopes or purification tags, for example: β-galactosidase,glutathione-S-transferase, green fluorescent proteins (GFP), epitopetags such as FLAG, myc tag, poly histidine, or viral surface proteinssuch as influenza virus haemagglutinin, or bacterial proteins such astetanus toxoid, diphtheria toxoid, CRM197.

Proteins

In an embodiment, the immunogenic composition of the invention furthercomprises one or more of the proteins mentioned below. Many of thepreferred proteins fall into the categories of extracellular componentbinding proteins, transporter proteins or toxins and regulators ofvirulence. The immunogenic composition of the invention optionallyfurther comprises a staphylococcal extracellular component bindingprotein or a. staphylococcal transporter protein or a staphylococcaltoxin or regulator of virulence. The immunogenic composition of theinvention optionally comprises at least or exactly 1, 2, 3, 4, 5 or 6staphylococcal proteins.

TABLE 1 The following table sets out the SEQ ID numbers of proteinsequences and DNA sequences that are found in FIG. 1 and FIG. 2respectively. Name Protein sequence DNA sequence Immunodominant ABCtransporter SA SEQ ID 1 SEQ ID 34 SE SEQ ID 2 SEQ ID 35 Laminin receptorSA SEQ ID 3 SEQ ID 36 SE SEQ ID 4 SEQ ID 37 Secretory Antigen A SsaA SA1 SEQ ID 5 SEQ ID 38 SA 2 SEQ ID 6 SEQ ID 39 SE SEQ ID 7 SEQ ID 40 SitCSA SEQ ID 8 SEQ ID 41 SE SEQ ID 9 SEQ ID 42 IsaA/PisA (IssA) SA SEQ ID10 SEQ ID 43 SE SEQ ID 11 SEQ ID 44 EbhA/B SA EbhA SEQ ID 12 SEQ ID 45SA EbhB SEQ ID 13 SEQ ID 46 SE EbhA SEQ ID 14 SEQ ID 47 SE EbhB SEQ ID15 SEQ ID 48 Accumulation-assoc pro Aap SA SEQ ID 16 SEQ ID 49 SE SEQ ID17 SEQ ID 50 RNA III activating protein RAP SA SEQ ID 18 SEQ ID 51 SESEQ ID 19 SEQ ID 52 FIG/SdrG SA SEQ ID 20 SEQ ID 53 SE SEQ ID 21 SEQ ID54 Elastin binding protein EbpS SA SEQ ID 22 SEQ ID 55 SE SEQ ID 23 SEQID 56 Extracellular protein EFB SA SEQ ID 24 SEQ ID 57 alpha toxin SASEQ ID 25 SEQ ID 58 SBI SA SEQ ID 26 SEQ ID 59 IsdA SA SEQ ID 27 SEQ ID60 IsdB SA SEQ ID 28 SEQ ID 61 SdrC SA SEQ ID 29 SEQ ID 62 ClfA SA SEQID 30 SEQ ID 63 FnbA SA SEQ ID 31 SEQ ID 64 ClfB SA SEQ ID 32 SEQ ID 65Coagulase SA SEQ ID 33 SEQ ID 66 FnbB SA SEQ ID 67 SEQ ID 71 MAP SA SEQID 68 SEQ ID 72 SdrC SA SEQ ID 69 SEQ ID 73 SdrG SA SEQ ID 70 SEQ ID 74SA indicates a sequence from S. aureus and SE indicates a sequence fromS. epidermidis.

Extracellular Component Binding Proteins

Extracellular component binding proteins are proteins that bind to hostextracellular components. The term includes, but is not limited toadhesins.

Examples of extracellular component binding proteins include lamininreceptor (Naidu et al J. Med. Microbiol. 1992, 36; 177),SitC/MntC/saliva binding protein (U.S. Pat. No. 5,801,234, Wiltshire andFoster Infec. Immun. 2001, 69; 5198), EbhA (Williams et al Infect.Immun. 2002, 70; 6805), EbhB, Elastin binding protein (EbpS) (Park et al1999, J. Biol. Chem. 274; 2845), EFB (FIB) (Wasffelt and Flock 1995, J.Clin. Microbiol. 33; 2347), SBI (Zhang et al FEMS Immun. Med. Microbiol.2000, 28; 211), autolysin (Rupp et al 2001, J. Infect. Dis. 183; 1038),ClfA (U.S. Pat. No. 6,008,341, McDevitt et al Mol. Microbiol. 1994, 11;237), SdrC, SdrG (McCrea et al Microbiology 2000, 146; 1535), SdrH(McCrea et al Microbiology 2000, 146; 1535), Lipase GehD(US2002/0169288), SasA, FnbA (Flock et al Mol Microbiol. 1994, 12; 599,U.S. Pat. No. 6,054,572), FnbB (WO 97/14799, Booth et al 2001 Infec.Immun. 69; 345), collagen binding protein Cna (Visai et al 2000, J.Biol. Chem. 275; 39837), ClfB (WO 99/27109), FbpA (Phonimdaeng et al1988 J. Gen Microbio1.134; 75), Npase (Flock 2001 J. Bacteriol. 183;3999), IsaA/PisA (Lonenz et al FEMS Immuno. Med. Microbiol. 2000, 29;145), SsaA (Lang et al FEMS Immunol. Med. Microbiol. 2000, 29; 213), EPB(Hussain and Hermann symposium on Staph Denmark 14-17^(th) 2000), SSP-1(Veenstra et al 1996, J. Bacteriol. 178; 537), SSP-2 (Veenstra et al1996, J. Bacteriol. 178; 537), 17 kDa heparin binding protein HBP(Fallgren et al 2001, J. Med. Microbiol. 50; 547), Vitronectin bindingprotein (Li et al 2001, Curr. Microbiol. 42; 361), fibrinogen bindingprotein, coagulase, Fig (WO 97/48727) and MAP (U.S. Pat. No. 5,648,240)

SitC/MntC/Saliva Binding Protein

This is an ABC transporter protein which is a homologue of adhesin PsaAin S. pneumoniae. It is a highly immunogenic 32 kDa lipoprotein which isdistributed through the bacterial cell wall (Cockayne et al Infect.Immun. 1998 66; 3767). It is expressed in S. aureus and S. epidermidisas a 32 kDa lipoprotein and a 40 kDa homologue is present in S. hominis.In S. epidermidis, it is a component of an iron-regulated operon. Itshows considerable homology to both adhesins including FimA ofStreptococcus parasanguis, and with lipoproteins of a family of ABCtransporters with proven or putative metal iron transport functions.Therefore SitC is included as an extracellular biding protein and as ametal ion transporter.

The saliva binding protein disclosed in U.S. Pat. No. 5,801,234 is alsoa form of SitC and can be included in an immunogenic composition of theinvention.

ClfA and ClfB

Both these proteins have fibrinogen binding activity and trigger S.aureus to form clumps in the presence of plasma. They contain a LPXTGmotif common to wall associated proteins.

ClfA is described in U.S. Pat. No. 6,008,341 and ClfB is described in WO99/27109.

Coaqulase (FbpA)

This is a fibrinogen binding protein which triggers S. aureus to formclumps in the presence of plasma. It is described in references relatedto Coagulase: Phonimdaeng et al (J. Gen. Microbio. 1988, 134:75-83),Phonimdaeng et al. (Mol Microbiol 1990; 4:393-404), Cheung et al.(Infect Immun 1995; 63:1914-1920) and Shopsin et al. (J. CLin.Microbiol. 2000; 38:3453-3456).

Preferred fragments for inclusion in the immunogenic composition of theinvention include the mature protein in which the signal peptide hasbeen removed (amino acids 27 to the C-terminus).

Coagulase has three distinct domains. Amino acids 59-297 which is acoiled coil region, amino acids 326-505 which is a proline and glycinerich region and the C-terminal domain from amino acid 506 to 645 whichhas a beta sheet conformation. Each of these domains is a fragment whichmay be incorporated into the immunogenic composition of the invention.

SdrG

This protein is described in WO 00/12689. SdrG is found in coagulasenegative staphylococci and is a cell wall associated protein containinga LPXTG sequence.

SdrG contains a signal peptide (amino acids 1-51), a region containingfibrinogen binding sites and collagen binding sites (amino acids51-825), two CnaB domains (amino acids 627-698 and 738-809), a SD repeatregion (amino acids 825-1000) and an anchor domain (amino acids1009-1056).

Preferred fragments of SdrG include polypeptides in which the signalpeptide and/or the SD repeats and the anchor domain have been removed.These include polypeptides comprising or consisting of amino acids50-825, amino acids 50-633, amino acids 50-597 (SEQ ID NO 2 of WO03/76470), amino acids 273-597 (SEQ ID NO 4 of WO 03/76470), amino acids273-577 (SEQ ID NO 6 of WO 03/76470) amino acids 1-549, amino acids219-549, amino acids 225-549, amino acids 219-528, amino acids 225-528of SEQ ID NO: 70 or 20 or 21.

Preferably, an SdrG polypeptide having a sequence at least 80%, 85%,90%, 92%, 95%, 97%, 98%, 99% or 100% homologous to the sequence of SEQID NO: 70, 20 or 21 is incorporated into the immunogenic composition ofthe invention.

The compositions of the invention optionally comprise a fragment of theSdrG polypeptides described above.

Preferred fragments have the signal peptide and/or the SD repeat domainand/or the anchoring domain deleted. For example sequences correspondingto amino acids 1-713, 1-549, 225-549, 225-529, 24-717, 1-707, 1-690,1-680, 1-670, 1-660, 1-650, 1-640, 1-630, 1-620, 1-610, 1-600, 34-707,44-697, 36-689 of SEQ ID 70 or sequences having 85%, 90%, 92%, 95%, 97%,98%, 99% or 100% identity to SEQ ID 70 or 20 or 21.

Preferred fragments with the signal peptide deleted have a methionineresidue at the N-terminus of the fragment to ensure correct translation.

A more preferred fragment has the following sequence:—

MEENSVQDVKDSNTDDELSDSNDQSSDEEKNDVINNNQSINTDDNNQIIKKEETNNYDGIEKRSEDRTESTTNVDENEATFLQKTPQDNTHLTEEEVKESSSVESSNSSIDTAQQPSHTTINREESVQTSDNVEDSHVSDFANSKIKESNTESGKEENTIEQPNKVKEDSTTSQPSGYTNIDEKISNQDELLNLPINEYENKARPLSTTSAQPSIKRVTVNQLAAEQGSNVNHLIKVTDQSITEGYDDSEGVIKAHDAENLIYDVTFEVDDKVKSGDTMTVDIDKNTVPSDLTDSFTIPKIKDNSGEIIATGTYDNKNKQITYTFTDYVDKYENIKAHLKLTSYIDKSKVPNNNTKLDVEYKTALSSVNKTITVEYQRPNENRTANLQSMFTNIDTKNHTVEQTIYINPLRYSAKETNVNISGNGDEGSTIIDDSTIIKVYKVGDNQNLPDSNRIYDYSEYEDVTNDDYAQLGNNNDVNINFGNIDSPYIIKVISKYDPNKDDYTTIQQTVTMQTTINEYTGEFRTASYDNTIAFSTSSGQGQGDLPPEKTYKIGDYVWEDVDKDGIQNTNDNEKPLSNVLVTLTYPDGTSKSVRTDEDGKYQFDGLKNGLTYKITFETPEGYTPTLKHSGTNPALDSEGNSVWVTINGQDDMTIDSGFYQTPKYSLGNYVWDTNKDGIQGDDEKGISGVKVTLKDENGNIISTTTTDENGKYQFDNLNSGNYIVHFDKPSGMTQTTTDSGDDDEQDADGEEVHVTITDHDDFSIDNGYY DDE

EbhA and EbhB

EbhA and EbhB are proteins that are expressed in both S. aureus and S.epidermidis (Clarke and Foster Infect. Immun. 2002, 70; 6680, Williamset al Infect. Immun. 2002, 20; 6805) and which bind to fibronectin.Since fibronectin is an important component of extracellular matrix,EbhA and EbhB have an important function in adhering staphylococci tohost extracellular matrix.

The Ebh proteins are large, having a molecular weight of 1.1megadaltons. It is advantageous to use a fragment of the Ebh proteinrather than the complete sequence due to ease of production andformulation. The central region of the protein contains imperfectrepeats which contain fibronectin binding sites. Fragments containingone or more of the repeat domains described below are preferredfragments for incorporation into the immunogenic composition of theinvention.

Ebh proteins contain imperfect repeats units of 127 amino acids inlength which are characterised by containing the consensus sequence:—

L.G. {10}A. {13}Q. {26}L...M..L. {33}A Preferably . {19}L.G. {10}A.{13}Q. {26}L...M..L. {33}A. {12} More Preferably.....I/V..A...I/V..AK.ALN/DG..NL..AK..A. {6}L..LN.AQK..L..QI/V..A..V..V.{6}A..LN/D.AM..L...I/V.D/E...TK.S.NY/F.N/DAD.K.AY/F.AV..A..I/V.N/D.......

Where ‘.’ means any amino acid and ‘.{10}’ means any 10 amino acids andI/V indicates alternative choices of amino acid.

By reference to the sequence disclosed in Kuroda et al (2001) Lancet357; 1225-1240, and Table 2, the repeat sequences within Ebh proteinsare readily deduced.

Preferred fragments to be included in the immunogenic composition of theinvention include proteins containing of one, two, three, four, five,six, seven, eight, nine, ten or more than 10 of the 127 amino acidrepeat units. Such fragments may consist of 1, 2, 3, 4, 5, 6, 7, 8, 9,10 or more repeats of the 127 amino acid repeat region or may consist of1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more repeats with additional amino acidresidues present at either or both ends of the fragment. A furtherpreferred fragment is the H2 polypeptide of about 44 kDa spanning threerepeats (amino acids 3202-3595) as described in Clarke et al Infectionand Immunity 70, 6680-6687, 2002. Such fragments will preferably be ableto bind fibronectin and/or to elicit antibodies that are reactiveagainst the whole Ebh protein.

The Ebh proteins are capable of binding to fibronectin. Preferredfragments of these polypeptides sequences retain the ability to bind tofibronectin. Binding to fibronectin can be assessed by ELISA asdescribed by Clarke et al (Infection and Immunity 70; 6680-6687 2002).

Still further preferred fragments are those which comprise a B-cell orT-helper epitope, for example those fragments/peptides described inTables 3 and 4.

TABLE 2 Repeat sequences in the full-length sequence of Ebh. Thefull-length sequence of Ebh is disclosed in Kuroda et al (2001) Lancet357; 1225-1240. The following table shows the amino acid residues atwhich the 127 amino acid repeats begin and end within the full lengthsequence. Begin End 1 3204 3330 2 3331 3457 3 3457 3583 4 3583 3709 53709 3835 6 3835 3961 7 3961 4087 8 4200 4326 9 4326 4452 10 4452 457811 4578 4704 12 4704 4830 13 4830 4956 14 4956 5082 15 5082 5208 16 52085334 17 5334 5460 18 5460 5586 19 5585 5711 20 5711 5837 21 5837 5963 225963 6089 23 6089 6215 24 6215 6341 25 6341 6467 26 6467 6593 27 65936719 28 6719 6845 29 6845 6971 30 6971 7097 31 7097 7223 32 7223 7349 337349 7475 34 7475 7601 35 7601 7727 36 7727 7853 37 7852 7978 38 79788104 39 8104 8230 40 8230 8356 41 8356 8482 42 8482 8608 43 8604 8730 448858 8984

TABLE 3 B-cell epitope prediction for a 127 amino acid  repeat:The full-length sequence is disclosed in Kurodaet al (2001) Lancet 357; 1225-1240. One of theserepeats, encoded by amino acids 3204-3331 of thefull-length sequence was chosen to carry out an epitope prediction:-MDVNTVNQKAASVKSTKDALDGQQNLQRAKTEATNAITHASDLNQAQKNALTQQVNSAQNVHAVNDIKQTTQSLNTAMTGLKRGVANHNQVVQSDNYVNADTNKKNDYNNAYNHANDIINGNAQHPVI Begin End Epitope sequence Start Stop 510 TVNQKA 3208 3213 14 19 KSTKDA 3217 3222 21 33 DGQQNLQRAKTEA 3224 323642 51 DLNQAQKNAL 3245 3254 66 74 DIKQTTQSL 3269 3277 100 112ADTNKKNDYNNAY 3303 3315 117 123 DIINGNA 3320 3326 The “Begin” and “End”columns present the position of the predicted B-cell epitopes in the 127amino acid repeat The “Start” and “Stop” columns present the position ofthe predicted B-cell epitopes in the Ebh full length sequence

TABLE 4 T-helper cell epitope prediction in Ebh:The full-length sequence is disclosed in TrEMBLdatabase, sequence reference Q8NWQ6. One ofthese repeats, encoded by amino acids 3204-3331of the full-length sequence was chosen to carryout an epitope prediction:-MDVNTVNQKAASVKSTKDALDGQQNLQRAKTEATNAITHASDLNQAQKNALTQQVNSAQNVHAVNDIKQTTQSLNTAMTGLKRGVANHNQVVQSDNYVNADTNKKNDYNNAYNHANDIINGNAQHPVI Position Position repeatEpitope sequence sequence 1 MDVNTVNQK 3204 3 VNTVNQKAA 3206 6 VNQKAASVK3209 26 LQRAKTEAT 3229 37 ITHASDLNQ 3240 43 LNQAQKNAL 3246 51 LTQQVNSAQ3254 55 VNSAQNVHA 3258 61 VHAVNDIKQ 3264 64 VNDIKQTTQ 3267 67 IKQTTQSLN3270 74 LNTAMTGLK 3277 78 MTGLKRGVA 3281 81 LKRGVANHN 3284 85 VANHNQVVQ3288 91 VVQSDNYVN 3294 92 VQSDNYVNA 3295 97 YVNADTNKK 3301 98 VNADTNKKN3302 108 YNNAYNHAN 3311 112 YNHANDIIN 3315 118 IINGNAQHP 3321 119INGNAQHPV 3322 The “Position repeat” column presents the position of thepredicted T-cell epitopes in the repeat The “Position sequence” columnpresents the position of the predicted T-cell epitopes the repeat in theEbh full length sequence

Fragments of the proteins of the invention may be employed for producingthe corresponding full-length polypeptide by peptide synthesis;therefore, these fragments may be employed as intermediates forproducing the full-length proteins of the invention.

Particularly preferred are variants in which several, 5-10, 1-5, 1-3,1-2 or 1 amino acids are substituted, deleted, or added in anycombination.

Elastin Binding Protein (EbpS)

EbpS is a protein containing 486 amino acids with a molecular weight of83 kDa. It is associated with the cytoplasmic membrane of S. aureus andhas three hydrophobic regions which hold the protein in the membrane(Downer et al 2002, J. Biol. Chem. 277; 243; Park et al 1996, J. Biol.Chem. 271; 15803).

Two regions between amino acids 1-205 and 343-486 are surface exposed onthe outer face of the cytoplasmic membrane. The ligand binding domain ofEbpS is located between residues 14-34 at the N-terminus (Park et al1999, J. Biol. Chem. 274; 2845).

A preferred fragment to be incorporated into the immunogenic compositionof the invention is the surface exposed fragment containing the elastinbinding region (amino acids 1-205). Some preferred fragments do notcontain the entire exposed loop but should contain the elastin bindingregion (amino acids 14-34). An alternative fragment which could be usedconsists of amino acids forming the second surface exposed loop (aminoacids 343-486). Alternative fragments containing up to 1, 2, 5, 10, 20,50 amino acids less at one or both ends are also possible.

Laminin Receptors

The laminin receptor of S. aureus plays an important role inpathogenicity. A characteristic feature of infection is bloodstreaminvasion which allows widespread metastatic abscess formation.Bloodstream invasion requires the ability to extravasate across thevascular basement membrane. This is achieved through binding to lamininthrough the laminin receptor (Lopes et al Science 1985, 229; 275).

Laminin receptors are surface exposed and are present in many strains ofstaphylococci including S. aureus and S. epidermidis.

SBI

Sbi is a second IgG binding protein in addition to protein A and it isexpressed in most strains of S. aureus (Zhang et al 1998, Microbiology144; 985).

The N-terminus of the sequence of Sbi has a typical signal sequence witha cleavage site after amino acid 29. Therefore a preferred fragment ofSbi to be incorporated into an immunogenic composition of the inventionstarts at amino acid residue 30, 31, 32 or 33 and continues to theC-terminus of Sbi, for example of SEQ ID NO: 26.

The IgG binding domain of Sbi has been identified as a region towardsthe N-terminus of the protein from amino acids 41-92. This domain ishomologous to the IgG binding domains of protein A.

The minimal IgG binding domain of Sbi contains the following sequence:—

QTTQNNYVTDQQKAFYQVLHLKGITEEQRNQYIKTLREHPERAQ          ** ***  *        ***  *  *   * EVFSESLK     *  **-denotes amino acids which are similar between IgG binding domains

Preferred fragment of Sbi to be included in the immunogenic compositionof the invention contains an IgG binding domain. This fragment containsthe consensus sequence for an IgG binding domain as designated by * asshown in the above sequence. Preferably the fragment contains orconsists of the complete sequence shown above. More preferably, thefragment contains or consists of amino acids 30-92, 33-92, 30-94, 33-94,30-146, 33-146, 30-150, 33-150, 30-160, 33-160, 33-170, 33-180, 33-190,33-200, 33-205 or 33-210 of Sbi, for example of SEQ ID NO:26.

Preferred fragment may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acidsubstitutions from the sequences indicated.

Preferred fragments may contain multiple repeats (2, 3, 4, 5, 6, 7, 8, 9or 10) of the IgG binding domain.

EFB-FIB

Fib is a 19 kDa fibrinogen binding protein which is secreted into theextracellular medium by S. aureus. It is produced by all S aureusisolates tested (Wastfelt and Flock 1995, J. Clin. Microbiol. 33; 2347).

S. aureus clumps in the presence of fibrinogen and binds to fibrinogencoated surfaces. This ability facilitates staphylococcal colonisation ofcatheters and endothelial cells.

Fib contains a signal sequence at the N-terminus of the protein with aputative cleavage site at about amino acid 30. Therefore a preferredfragment to be introduced in the immunogenic composition of theinvention would contain the sequence of the mature protein (from aboutamino acid 30 to the C-terminus of the protein).

Fbe-EfB/FIG

Fbe is a fibrinogen binding protein that is found in many isolates of S.epidermidis and has a deduced molecular weight of 119 kDa (Nilsson et al1998. Infect. Immun. 66; 2666). Its sequence is related to that ofclumping factor from S. aureus (ClfA). Antibodies against Fbe can blockthe binding of S. epidermidis to fibrinogen coated plates and tocatheters (Pei and Flock 2001, J. Infect. Dis. 184; 52).

Fbe has a putative signal sequence with a cleavage site between aminoacids 51 and 52. Therefore a preferred fragment of Fbe contains themature form of Fbe extending from amino acid 52 to the C-terminus (aminoacid 1,092).

The domain of Fbe from amino acid 52 to amino acid 825 is responsiblefor fibrinogen binding. Therefore a preferred fragment of Fbe consistsof or contains amino acids 52-825.

The region between amino acid 373 and 516 of Fbe shows the mostconservation between Fbe and ClfA. Preferred fragment will thereforecontain amino acid's 373-516 of Fbe.

Amino acids 825-1041 of Fbe contains a highly repetitive region composedof tandemly repeated aspartic acid and serine residues.

IsaA/PisA

IsaA is a 29 kDa protein, also known as PisA has been shown to be aimmunodominant staphylococcal protein during sepsis in hospital patients(Lorenz et al 2000, FEMS Immunol. Med. Microb. 29; 145).

The first 29 amino acids of the IsaA sequence are thought to be a signalsequence. Therefore a preferred fragment of IsaA to be included in animmunogenic composition of the invention would contain amino acidresidues 30 onwards, to the end of the coded sequence.

Fibronectin Binding Protein

Fibronectin binding protein A contains several domains that are involvedin binding to fibronectin (WO 94/18327). These are called D1, D2, D3 andD4. Preferred fragments of fibronectin binding protein A or B compriseor consist of D1, D2, D3, D4, D1-D2, D2-D3, D3-D4, D1-D3, D2-D4 orD1-D4.

Fibronectin binding protein contains a 36 amino acid signal sequence.For example:

VKNNLRYGIRKHKLGAASVFLGTMIVVGMGQDKEAA

Optionally, the mature protein omitting this signal sequence is includedin the immunogenic composition of the invention.

Transporter Proteins

The cell wall of Gram positive bacteria acts as a barrier preventingfree diffusion of metabolites into the bacterium. A family of proteinsorchestrates the passage of essential nutrients into the bacterium andare therefore essential for the viability of the bacterium. The termtransporter protein covers proteins involved in the initial step ofbinding to metabolites such as iron as well as those involved inactually transporting the metabolite into the bacterium.

Molecular iron is an essential co-factor for bacterial growth.Siderophores are secreted that bind free iron and then are captured bybacterial surface receptors that deliver iron for transport across thecytoplasmic membrane. Iron acquisition is critical for the establishmentof human infections so that the generation of an immune response againstthis class of proteins leads to a loss of staphylococcal viability.

Examples of transporter proteins include Immunodominant ABC transporter(Burnie et al 2000 Infect. Imun. 68; 3200), IsdA (Mazmanian et al 2002PNAS 99; 2293), IsdB (Mazmanian et al 2002 PNAS 99; 2293), Mg2+transporter, SitC (Wiltshire and Foster 2001 Infect. Immun. 69; 5198)and Ni ABC transporter.

Immunodominant ABC Transporter

Immunodominant ABC transporter is a well conserved protein which may becapable of generating an immune response that is cross-protectiveagainst different staphylococcal strains (Mei et al 1997, Mol.Microbiol. 26; 399). Antibodies against this protein have been found inpatients with septicaemia (Burnie et al 2000, Infect. Immun. 68; 3200).

Preferred fragments of immunodominant ABC transporter will include thepeptides DRHFLN, GNYD, RRYPF, KTTLLK, GVTTSLS, VDWLR, RGFL, morepreferably KIKVYVGNYDFWYQS, TVIVVSHDRHFLYNNV and/or TETFLRGFLGRMLFSsince these sequences contain epitopes that are recognised by the humanimmune system.

IsdA-IsdB

The isd genes (iron-regulated surface determinant) of S. aureus encodeproteins responsible for haemoglobin binding and passage of haem iron tothe cytoplasm, where it acts as an essential nutrient. IsdA and IsdB arelocated in the cell wall of staphylococci. IsdA appear to be exposed onthe surface of bacterium since it is susceptible to proteinase Kdigestion. IsdB was partially digested suggesting that it is partiallyexposed on the surface of the bacterium (Mazmanian et al 2003 Science299; 906).

IsdA and IsdB are both 29 kDa proteins which bind heme. Their expressionis regulated by the availability of iron via the Fur repressor. Theirexpression will be high during infection in a host where theconcentration of iron will be low.

They are also known as FrpA and FrpB (Morrissey et al 2002, Infect.Immun. 70; 2399). FrpA and FrpB are major surface proteins with a highcharge. They have been shown to provide a major contribution to adhesionto plastic.

In an embodiment, the immunogenic composition of the invention comprisesa fragment of IsdA and/or IsdB which is described in WO 01/98499 or WO03/11899.

Toxins and Regulators of Virulence

Members of this family of proteins include toxin such as alpha toxin,hemolysin, enterotoxin B and TSST-1 as well as proteins that regulatethe production of toxins such as RAP.

Alpha Toxin (Hla)

Alpha toxin is an important virulence determinant produced by moststrains of S. aureus. It is a pore forming toxin with haemolyticactivity. Antibodies against alpha toxin have been shown to neutralisethe detrimental and lethal effects of alpha toxin in animal models(Adlam et al 1977 Infect. Immun. 17; 250). Human platelets, endothelialcells and mononuclear cells are susceptible to the effects of alphatoxin.

The high toxicity of alpha toxin requires that it should be detoxifiedbefore being used as an immunogen. This can be achieved by chemicaltreatment, for instance by treating with formaldehyde, glutaraldehyde ofother cross-linking reagents or by chemically conjugating it tobacterial polysaccharides or to LTA as described below.

A further way of removing toxicity is to introduce point mutations thatremove toxicity while retaining the antigenicity of the toxin. Theintroduction of a point mutation at amino acid 35 of alpha toxin where ahistidine residue is replaced with a leucine residue results in theremoval of toxicity whilst retaining immunogenicity (Menzies andKernodle 1996; Infect. Immun. 64; 1839). Histidine 35 appears to becritical for the proper oligomerization required for pore formation andmutation of this residue leads to loss of toxicity.

When incorporated into immunogenic compositions of the invention, alphatoxin is preferably detoxified by mutation of His 35, most preferably byreplacing His 35 with Leu or Arg. In an alternative embodiment, alphatoxin is detoxified by conjugation to other components of theimmunogenic composition, preferably capsular polysaccharides or LTA,most preferably to S. aureus type V polysaccharide and/or S. aureus TypeVIII polysaccharide and/or PIA.

RNA III Activating Protein (RAP)

RAP is not itself a toxin, but is a regulator of the expression ofvirulence factors. RAP is produced and secreted by staphylococci. Itactivates the agr regulatory system of other staphylococci and activatesthe expression and subsequent release of virulence factors such ashemolysin, enterotoxin B and TSST-1.

An immune response generated against RAP would not kill the bacteriumbut would interfere with their pathogenicity. This has the advantage ofproviding less selective pressure for new resistant strains to emerge.

It would have a second advantage of producing an immune response thatwould be instrumental in reducing the morbidity of the infection.

It is particularly advantageous to combine RAP with other antigens in avaccine, particularly where the additional antigen would provide animmune response that is able to kill the bacterium.

Other Immunodominant Proteins Accumulation-Associated Protein (Aap)

Aap is a 140 kDa protein which is essential for the accumulation of S.epidermidis strains on surfaces (Hussain et al Infect. Immun. 1997, 65;519). Strains expressing this protein produced significantly largeramounts of biofilm and Aap appear to be involved in biofilm formation.Antibodies against Aap are able to inhibit biofilm formation and inhibitthe accumulation of S. epidermidis.

Staphylococcal Secretory Antigen SsaA

SsaA is a strongly immunogenic protein of 30 kDa found in both S. aureusand S. epidermidis (Lang et al 2000 FEMS Immunol. Med. Microbiol. 29;213). Its expression during endocarditis suggested a virulence rolespecific to the pathogenesis of the infectious disease.

SsaA contains an N-terminal leader sequence and a signal peptidasecleavage site. The leader peptide is followed by a hydrophilic region ofapproximately 100 amino acids from residue 30 to residue 130.

A preferred fragment of SsaA to be incorporated into the immunogeniccomposition of the invention is made up of the mature protein (aminoacids 27 to the C-terminus or amino acids 30 to the C-terminus).

A further preferred fragments contains the hydrophilic area of SsaA fromamino acid 30 to amino acid 130.

Preferred Combinations

Staphylococcal infections progress through several different stages. Forexample, the staphylococcal life cycle involves commensal colonisation,initiation of infection by accessing adjoining tissues or thebloodstream, anaerobic multiplication in the blood, interplay between S.aureus virulence determinants and the host defense mechanisms andinduction of complications including endocarditis, metastatic abscessformation and sepsis syndrome. Different molecules on the surface of thebacterium will be involved in different steps of the infection cycle. Bytargeting the immune response against a combination of particularantigens involved in different processes of Staphylococcal infection,multiple aspects of staphylococcal function are affected and this canresult in good vaccine efficacy.

In particular, combinations of certain antigens from different classes,some of which are involved in adhesion to host cells, some of which areinvolved in iron acquisition or other transporter functions, some ofwhich are toxins or regulators of virulence and immunodominant antigenscan elicit an immune response which protects against multiple stages ofinfection.

Some combinations of antigens are particularly effective at inducing animmune response. This can be measured either in animal model assays asdescribed in the examples and/or using an opsonophagocytic assay asdescribed in the examples. Without wishing to be bound by theory, sucheffective combinations of antigens are thought to be enabled by a numberof characteristics of the immune response to the antigen combination.The antigens themselves are usually exposed on the surface ofStaphylococcal cells, they tend to be conserved but also tend not to bepresent in sufficient quantity on the surface cell for an optimalbactericidal response to take place using antibodies elicited againstthe single antigen. Combining the antigens of the invention can resultin a formulation eliciting an advantageous combination of antibodieswhich interact with the Staphylococcal cell beyond a critical threshold.At this critical level, sufficient antibodies of sufficient quality bindto the surface of the bacterium to allow either efficient killing bycomplement or neutralisation of the bacterium. This can be measured ineither an animal challenge model or an opsonisation assay as describedin the examples.

Preferred immunogenic compositions of the invention comprise a pluralityof proteins selected from at least two different categories of protein,having different functions within Staphylococci. Examples of suchcategories of proteins are extracellular binding proteins, transporterproteins such as Fe acquisition proteins, toxins or regulators ofvirulence and other immunodominant proteins.

In a preferred embodiment, the immunogenic composition of the inventionfurther comprises a number of proteins equal to or greater than 2, 3, 4,5 or 6 selected from 2 or 3 different groups selected from;

-   -   Group a) extracellular component binding proteins;    -   Group b) transporter proteins;    -   Group c) toxins or regulators of virulence.

In a preferred embodiment, the immunogenic composition of the inventionfurther comprises a number of proteins equal to or greater than 2, 3, 4,5 or 6 selected from 2 or 3 of the following groups:

-   -   group a)—at least one staphylococcal extracellular component        binding protein or fragment thereof selected from the group        consisting of laminin receptor, SitC/MntC/saliva binding        protein, EbhA, EbhB, Elastin binding protein (EbpS), EFB (FIB),        SBI, autolysin, ClfA, SdrC, SdrG, SdrH, Lipase GehD, SasA, FnbA,        FnbB, Cna, ClfB, FbpA, Npase, IsaA/PisA, SsaA, EPB, SSP-1,        SSP-2, HBP, Vitronectin binding protein, fibrinogen binding        protein, coagulase, Fig and MAP;    -   group b)—at least one staphylococcal transporter protein or        fragment thereof selected from the group consisting of        Immunodominant ABC transporter, IsdA, IsdB, Mg2+ transporter,        SitC and Ni ABC transporter;    -   group c)—at least one staphylococcal regulator of virulence,        toxin or fragment thereof selected from the group consisting of        alpha toxin (Hla), alpha toxin H35R mutant, RNA III activating        protein (RAP).

In a preferred embodiment, the immunogenic composition of the inventioncontains at least one protein selected from group a) and an additionalprotein selected from group b) and/or group c).

In a further embodiment, the immunogenic composition of the inventioncontains at least one antigen selected from group b) and an additionalprotein selected from group c) and/or group a).

In a further embodiment, the immunogenic composition of the inventioncontains at least one antigen selected from group c) and an additionalprotein selected from group a) and/or group b).

A preferred combination of proteins in the immunogenic composition ofthe invention comprises laminin receptor and 1, 2, 3, 4 or 5 furtherantigens selected from the group consisting of immunodominant ABCtransporter, IsdA, IsdB, Mg2+ transporter, SitC, Ni ABC transporter,alpha toxin, alpha toxin H35L OR H35R mutant, RAP, Aap and SsaA.

A further preferred combination of proteins in the immunogeniccomposition of the invention comprises SitC and 1, 2, 3, 4 or 5 furtherantigens selected from the group consisting of immunodominant ABCtransporter, IsdA, IsdB, Mg2+ transporter, SitC, Ni ABC transporter,alpha toxin, alpha toxin H35L OR H35R mutant, RAP, Aap and SsaA.

A further preferred combination of proteins in the immunogeniccomposition of the invention comprises EbhA and 1, 2, 3, 4 or 5 furtherantigens selected from the group consisting of immunodominant ABCtransporter, IsdA, IsdB, Mg2+ transporter, SitC, Ni ABC transporter,alpha toxin, alpha toxin H35L OR H35R mutant, RAP, Aap and SsaA.

A further preferred combination of proteins in the immunogeniccomposition of the invention comprises EbhB and 1, 2, 3, 4 or 5 furtherantigens selected from the group consisting of immunodominant ABCtransporter, IsdA, IsdB, Mg2+ transporter, SitC, Ni ABC transporter,alpha toxin, alpha toxin H35L OR H35R mutant, RAP, Aap and SsaA.

A further preferred combination of proteins in the immunogeniccomposition of the invention comprises EbpS and 1, 2, 3, 4 or 5 furtherantigens selected from the group consisting of immunodominant ABCtransporter, IsdA, IsdB, Mg2+ transporter, SitC, Ni ABC transporter,alpha toxin, alpha toxin H35L OR H35R mutant, RAP, Aap and SsaA.

A further preferred combination of proteins in the immunogeniccomposition of the invention comprises EFB(FIB) and 1, 2, 3, 4 or 5further antigens selected from the group consisting of immunodominantABC transporter, IsdA, IsdB, Mg2+ transporter, SitC, Ni ABC transporter,alpha toxin, alpha toxin H35L OR H35R mutant, RAP, Aap and SsaA.

A further preferred combination of proteins in the immunogeniccomposition of the invention comprises SBI and 1, 2, 3, 4 or 5 furtherantigens selected from the group consisting of immunodominant ABCtransporter, IsdA, IsdB, Mg2+ transporter, SitC, Ni ABC transporter,alpha toxin, alpha toxin H35L OR H35R mutant, RAP, Aap and SsaA.

A further preferred combination of proteins in the immunogeniccomposition of the invention comprises autolysin and 1, 2, 3, 4 or 5further antigens selected from the group consisting of immunodominantABC transporter, IsdA, IsdB, Mg2+ transporter, SitC, Ni ABC transporter,alpha toxin, alpha toxin H35L OR H35R mutant, RAP, Aap and SsaA.

A further preferred combination of proteins in the immunogeniccomposition of the invention comprises ClfA and 1, 2, 3, 4 or 5 furtherantigens selected from the group consisting of immunodominant ABCtransporter, IsdA, IsdB, Mg2+ transporter, SitC, Ni ABC transporter,alpha toxin, alpha toxin H35L OR H35R mutant, RAP, Aap and SsaA.

A further preferred combination of proteins in the immunogeniccomposition of the invention comprises SdrC and 1, 2, 3, 4 or 5 furtherantigens selected from the group consisting of immunodominant ABCtransporter, IsdA, IsdB, Mg2+ transporter, SitC, Ni ABC transporter,alpha toxin, alpha toxin H35L OR H35R mutant, RAP, Aap and SsaA.

A further preferred combination of proteins in the immunogeniccomposition of the invention comprises SdrG and 1, 2, 3, 4 or 5 furtherantigens selected from the group consisting of immunodominant ABCtransporter, IsdA, IsdB, Mg2+ transporter, SitC, Ni ABC transporter,alpha toxin, alpha toxin H35L OR H35R mutant and RAP.

A further preferred combination of proteins in the immunogeniccomposition of the invention comprises SdrH and 1, 2, 3, 4 or 5 furtherantigens selected from the group consisting of immunodominant ABCtransporter, IsdA, IsdB, Mg2+ transporter, SitC, Ni ABC transporter,alpha toxin, alpha toxin H35L OR H35R mutant, RAP, Aap and SsaA.

A further preferred combination of proteins in the immunogeniccomposition of the invention comprises Lipase GehD and 1, 2, 3, 4 or 5further antigens selected from the group consisting of immunodominantABC transporter, IsdA, IsdB, Mg2+ transporter, SitC, Ni ABC transporter,alpha toxin, alpha toxin H35L OR H35R mutant, RAP, Aap and SsaA.

A further preferred combination of proteins in the immunogeniccomposition of the invention comprises SasA and 1, 2, 3, 4 or 5 furtherantigens selected from the group consisting of immunodominant ABCtransporter, IsdA, IsdB, Mg2+ transporter, SitC, Ni ABC transporter,alpha toxin, alpha toxin H35L OR H35R mutant, RAP, Aap and SsaA.

A further preferred combination of proteins in the immunogeniccomposition of the invention comprises FnbA and 1, 2, 3, 4 or 5 furtherantigens selected from the group consisting of immunodominant ABCtransporter, IsdA, IsdB, Mg2+ transporter, SitC, Ni ABC transporter,alpha toxin, alpha toxin H35L OR H35R mutant, RAP, Aap and SsaA.

A further preferred combination of proteins in the immunogeniccomposition of the invention comprises FnbB and 1, 2, 3, 4 or 5 furtherantigens selected from the group consisting of immunodominant ABCtransporter, IsdA, IsdB, Mg2+ transporter, SitC, Ni ABC transporter,alpha toxin, alpha toxin H35L OR H35R mutant, RAP, Aap and SsaA.

A further preferred combination of proteins in the immunogeniccomposition of the invention comprises Cna and 1, 2, 3, 4 or 5 furtherantigens selected from the group consisting of immunodominant ABCtransporter, IsdA, IsdB, Mg2+ transporter, SitC, Ni ABC transporter,alpha toxin, alpha toxin H35L OR H35R mutant, RAP, Aap and SsaA.

A further preferred combination of proteins in the immunogeniccomposition of the invention comprises ClfB and 1, 2, 3, 4 or 5 furtherantigens selected from the group consisting of immunodominant ABCtransporter, IsdA, IsdB, Mg2+ transporter, SitC, Ni ABC transporter,alpha toxin, alpha toxin H35L OR H35R mutant, RAP, Aap and SsaA.

A further preferred combination of proteins in the immunogeniccomposition of the invention comprises FbpA and 1, 2, 3, 4 or 5 furtherantigens selected from the group consisting of immunodominant ABCtransporter, IsdA, IsdB, Mg2+ transporter, SitC, Ni ABC transporter,alpha toxin, alpha toxin H35L OR H35R mutant, RAP, Aap and SsaA.

A further preferred combination of proteins in the immunogeniccomposition of the invention comprises Npase and 1, 2, 3, 4 or 5 furtherantigens selected from the group consisting of immunodominant ABCtransporter, IsdA, IsdB, Mg2+ transporter, SitC, Ni ABC transporter,alpha toxin, alpha toxin H35L OR H35R mutant, RAP, Aap and SsaA.

A further preferred combination of proteins in the immunogeniccomposition of the invention comprises IsaA/PisA and 1, 2, 3, 4 or 5further antigens selected from the group consisting of immunodominantABC transporter, IsdA, IsdB, Mg2+ transporter, SitC, Ni ABC transporter,alpha toxin, alpha toxin H35L OR H35R mutant, RAP, Aap and SsaA.

A further preferred combination of proteins in the immunogeniccomposition of the invention comprises SsaA and 1, 2, 3, 4 or 5 furtherantigens selected from the group consisting of immunodominant ABCtransporter, IsdA, IsdB, Mg2+ transporter, SitC, Ni ABC transporter,alpha toxin, alpha toxin H35L OR H35R mutant, RAP, Aap and SsaA.

A further preferred combination of proteins in the immunogeniccomposition of the invention comprises EPB and 1, 2, 3, 4 or 5 furtherantigens selected from the group consisting of immunodominant ABCtransporter, IsdA, IsdB, Mg2+ transporter, SitC, Ni ABC transporter,alpha toxin, alpha toxin H35L OR H35R mutant, RAP, Aap and SsaA.

A further preferred combination of proteins in the immunogeniccomposition of the invention comprises SSP-1 and 1, 2, 3, 4 or 5 furtherantigens selected from the group consisting of immunodominant ABCtransporter, IsdA, IsdB, Mg2+ transporter, SitC, Ni ABC transporter,alpha toxin, alpha toxin H35L OR H35R mutant, RAP, Aap and SsaA.

A further preferred combination of proteins in the immunogeniccomposition of the invention comprises SSP-2 and 1, 2, 3, 4 or 5 furtherantigens selected from the group consisting of immunodominant ABCtransporter, IsdA, IsdB, Mg2+ transporter, SitC, Ni ABC transporter,alpha toxin, alpha toxin H35L OR H35R mutant, RAP, Aap and SsaA.

A further preferred combination of proteins in the immunogeniccomposition of the invention comprises HPB and 1, 2, 3, 4 or 5 furtherantigens selected from the group consisting of immunodominant ABCtransporter, IsdA, IsdB, Mg2+ transporter, SitC, Ni ABC transporter,alpha toxin, alpha toxin H35L OR H35R mutant, RAP, Aap and SsaA.

A further preferred combination of proteins in the immunogeniccomposition of the invention comprises vitronectin binding protein and1, 2, 3, 4 or 5 further antigens selected from the group consisting ofimmunodominant ABC transporter, IsdA, IsdB, Mg2+ transporter, SitC, NiABC transporter, alpha toxin, alpha toxin H35L OR H35R mutant, RAP, Aapand SsaA.

A further preferred combination of proteins in the immunogeniccomposition of the invention comprises fibrinogen binding protein and 1,2, 3, 4 or 5 further antigens selected from the group consisting ofimmunodominant ABC transporter, IsdA, IsdB, Mg2+ transporter, SitC, NiABC transporter, alpha toxin, alpha toxin H35L OR H35R mutant, RAP, Aapand SsaA.

A further preferred combination of proteins in the immunogeniccomposition of the invention comprises coagulase and 1, 2, 3, 4 or 5further antigens selected from the group consisting of immunodominantABC transporter, IsdA, IsdB, Mg2+ transporter, SitC, Ni ABC transporter,alpha toxin, alpha toxin H35L OR H35R mutant, RAP, Aap and SsaA.

A further preferred combination of proteins in the immunogeniccomposition of the invention comprises Fig and 1, 2, 3, 4 or 5 furtherantigens selected from the group consisting of immunodominant ABCtransporter, IsdA, IsdB, Mg2+ transporter, SitC, Ni ABC transporter,alpha toxin, alpha toxin H35L OR H35R mutant, RAP, Aap and SsaA.

A further preferred combination of proteins in the immunogeniccomposition of the invention comprises MAP and 1, 2, 3, 4 or 5 furtherantigens selected from the group consisting of immunodominant ABCtransporter, IsdA, IsdB, Mg2+ transporter, SitC, Ni ABC transporter,alpha toxin, alpha toxin H35L OR H35R mutant, RAP, Aap and SsaA.

A further preferred combination of protein in the immunogeniccomposition of the invention comprises immunodominant ABC transporterand 1, 2, 3, 4 or 5 further antigens selected from the group consistingof laminin receptor, SitC/MntC/saliva binding protein, EbhA, EbhB,Elastin binding protein (EbpS), EFB (FIB), SBI, autolysin, ClfA, SdrC,SdrG, SdrH, Lipase GehD, SasA, FnbA, FnbB, Cna, ClfB, FbpA, Npase,IsaA/PisA, SsaA, EPB, SSP-1, SSP-2, HBP, Vitronectin binding protein,fibrinogen binding protein, coagulase, Fig, MAP, alpha toxin, alphatoxin H35L OR H35R mutant, RAP, Aap and SsaA.

A further preferred combination of protein in the immunogeniccomposition of the invention comprises IsdA and 1, 2, 3, 4 or 5 furtherantigens selected from the group consisting of laminin receptor,SitC/MntC/saliva binding protein, EbhA, EbhB, Elastin binding protein(EbpS), EFB (FIB), SBI, autolysin, ClfA, SdrC, SdrG, SdrH, Lipase GehD,SasA, FnbA, FnbB, Cna, ClfB, FbpA, Npase, IsaA/PisA, SsaA, EPB, SSP-1,SSP-2, HBP, Vitronectin binding protein, fibrinogen binding protein,coagulase, Fig, MAP, alpha toxin, alpha toxin H35L OR H35R mutant, RAP,Aap and SsaA.

A further preferred combination of protein in the immunogeniccomposition of the invention comprises IsdB and 1, 2, 3, 4 or 5 furtherantigens selected from the group consisting of laminin receptor,SitC/MntC/saliva binding protein, EbhA, EbhB, Elastin binding protein(EbpS), EFB (FIB), SBI, autolysin, ClfA, SdrC, SdrG, SdrH, Lipase GehD,SasA, FnbA, FnbB, Cna, ClfB, FbpA, Npase, IsaA/PisA, SsaA, EPB, SSP-1,SSP-2, HBP, Vitronectin binding protein, fibrinogen binding protein,coagulase, Fig, MAP, alpha toxin, alpha toxin H35L OR H35R mutant, RAP,Aap and SsaA.

A further preferred combination of protein in the immunogeniccomposition of the invention comprises SitC and 1, 2, 3, 4 or 5 furtherantigens selected from the group consisting of laminin receptor,SitC/MntC/saliva binding protein, EbhA, EbhB, Elastin binding protein(EbpS), EFB (FIB), SBI, autolysin, ClfA, SdrC, SdrG, SdrH, Lipase GehD,SasA, FnbA, FnbB, Cna, ClfB, FbpA, Npase, IsaA/PisA, SsaA, EPB, SSP-1,SSP-2, HBP, Vitronectin binding protein, fibrinogen binding protein,coagulase, Fig, MAP, alpha toxin, alpha toxin H35L OR H35R mutant, RAP,Aap and SsaA.

A further preferred combination of protein in the immunogeniccomposition of the invention comprises alpha toxin and 1, 2, 3, 4 or 5further antigens selected from the group consisting of laminin receptor,SitC/MntC/saliva binding protein, EbhA, EbhB, Elastin binding protein(EbpS), EFB (FIB), SBI, autolysin, ClfA, SdrC, SdrG, SdrH, Lipase GehD,SasA, FnbA, FnbB, Cna, ClfB, FbpA, Npase, IsaA/PisA, SsaA, EPB, SSP-1,SSP-2, HBP, Vitronectin binding protein, fibrinogen binding protein,coagulase, Fig, MAP, immunodominant ABC transporter, IsdA, IsdB, Mg2+transporter, SitC, Ni ABC transporter, Aap and SsaA.

A further preferred combination of protein in the immunogeniccomposition of the invention comprises alpha toxin H35L OR H35R variantand 1, 2, 3, 4 or 5 further antigens selected from the group consistingof laminin receptor, SitC/MntC/saliva binding protein, EbhA, EbhB,Elastin binding protein (EbpS), EFB (FIB), SBI, autolysin, ClfA, SdrC,SdrG, SdrH, Lipase GehD, SasA, FnbA, FnbB, Cna, ClfB, FbpA, Npase,IsaA/PisA, SsaA, EPB, SSP-1, SSP-2, HBP, Vitronectin binding protein,fibrinogen binding protein, coagulase, Fig, MAP, immunodominant ABCtransporter, IsdA, IsdB, Mg2+ transporter, SitC, Ni ABC transporter, Aapand SsaA.

A further preferred combination of protein in the immunogeniccomposition of the invention comprises RAP and 1, 2, 3, 4 or 5 furtherantigens selected from the group consisting of laminin receptor,SitC/MntC/saliva binding protein, EbhA, EbhB, Elastin binding protein(EbpS), EFB (FIB), SBI, autolysin, ClfA, SdrC, SdrG, SdrH, Lipase GehD,SasA, FnbA, FnbB, Cna, ClfB, FbpA, Npase, IsaA/PisA, SsaA, EPB, SSP-1,SSP-2, HBP, Vitronectin binding protein, fibrinogen binding protein,coagulase, Fig, MAP, immunodominant ABC transporter, IsdA, IsdB, Mg2+transporter, SitC, Ni ABC transporter, Aap and SsaA.

In the above and below combinations, the specified proteins mayoptionally be present in the immunogenic composition of the invention asa fragment or fusion protein as described above.

Combinations of Three Proteins

A preferred immunogenic composition of the invention further comprisesthree protein components in a combination of alpha-toxin, anextracellular component binding protein (preferably an adhesin) and atransporter protein (preferably an iron-binding protein).

In such a combination, the alpha toxin may be chemically detoxified orgenetically detoxified by introduction of point mutation(s), preferablythe His35Leu point mutation. The alpha toxin is present as a freeprotein or alternatively is conjugated to a polysaccharide or LTAcomponent of the immunogenic composition.

Preferred Combinations Include:—

An immunogenic composition comprising alpha toxin, IsdA and anextracellular component binding protein selected from the groupconsisting of laminin receptor, SitC/MntC/saliva binding protein, EbhA,EbhB, Elastin binding protein (EbpS), EFB (FIB), SBI, autolysin, ClfA,SdrC, SdrG, SdrH, Lipase GehD, SasA, FnbA, FnbB, Cna, ClfB, FbpA, Npase,IsaA/PisA, SsaA, EPB, SSP-1, SSP-2, HBP, Vitronectin binding protein,fibrinogen binding protein, coagulase, Fig and MAP.

An immunogenic composition comprising alpha toxin, IsdB and anextracellular component binding protein selected from the groupconsisting of laminin receptor, SitC/MntC/saliva binding protein, EbhA,EbhB, Elastin binding protein (EbpS), EFB (FIB), SBI, autolysin, ClfA,SdrC, SdrG, SdrH, Lipase GehD, SasA, FnbA, FnbB, Cna, ClfB, FbpA, Npase,IsaA/PisA, SsaA, EPB, SSP-1, SSP-2, HBP, Vitronectin binding protein,fibrinogen binding protein, coagulase, Fig and MAP.

An immunogenic composition comprising alpha toxin, IsdA and an adhesinselected from the group consisting of laminin receptor, EbhA, EbhB,Elastin binding protein (EbpS), EFB (FIB), ClfA, SdrC, SdrG, SdrH,autolysin, FnbA, FnbB, Cna, ClfB, FbpA, Npase, SSP-1, SSP-2, Vitronectinbinding protein, fibrinogen binding protein, coagulase, Fig and MAP.

An immunogenic composition comprising alpha toxin, IsdB and an adhesinselected from the group consisting of laminin receptor, EbhA, EbhB,Elastin binding protein (EbpS), EFB (FIB), autolysin, ClfA, SdrC, SdrG,SdrH, FnbA, FnbB, Cna, ClfB, FbpA, Npase, SSP-1, SSP-2, Vitronectinbinding protein, fibrinogen binding protein, coagulase, Fig and MAP.

An immunogenic composition comprising alpha toxin, IsdA and lamininreceptor.

An immunogenic composition comprising alpha toxin, IsdA and EbhA.

An immunogenic composition comprising alpha toxin, IsdA and EbhB.

An immunogenic composition comprising alpha toxin, IsdA and EbpS.

An immunogenic composition comprising alpha toxin, IsdA and EFB (FIB).

An immunogenic composition comprising alpha toxin, IsdA and SdrG.

An immunogenic composition comprising alpha toxin, IsdA and ClfA.

An immunogenic composition comprising alpha toxin, IsdA and ClfB.

An immunogenic composition comprising alpha toxin, IsdA and FnbA.

An immunogenic composition comprising alpha toxin, IsdA and coagulase.

An immunogenic composition comprising alpha toxin, IsdA and Fig.

An immunogenic composition comprising alpha toxin, IsdA and SdrH.

An immunogenic composition comprising alpha toxin, IsdA and SdrC.

An immunogenic composition comprising alpha toxin, IsdA and MAP.

An immunogenic composition comprising IsaA and Sbi.

An immunogenic composition comprising IsaA and IsdB.

An immunogenic composition comprising IsaA and IsdA.

An immunogenic composition comprising IsaA and SdrC.

An immunogenic composition comprising IsaA and Ebh or fragment thereofas described above.

An immunogenic composition comprising Sbi and SdrC.

An immunogenic composition comprising Sbi and Ebh or fragment thereof asdescribed above.

An immunogenic composition of the invention comprising IsaA, Sbi or SdrC

Selection of Antigens Expressed in Different Clonal Lineages

Analysis of the occurrence of virulence factors in relation with thepopulation structure of Staphylococcus aureus showed variable presenceof virulence genes in natural populations of S. aureus.

Among clinical isolates of Staphylococcus aureus, at least five clonallineages were shown to be highly prevalent (Booth et al., 2001 InfectImmun. 69(1):345-52). Alpha-hemolysin (hla), fibronectin-binding proteinA (fnbA) and clumping factor A (clfA) were shown to be present in mostof the isolates, regardless of lineage identity, suggesting an importantrole of these proteins in the survival of S. aureus (Booth et al., 2001Infect Immun. 69(1):345-52). Moreover, according to Peacock et al. 2002the distributions of fnbA, clfA, coagulase, spa, map, pvl(Panton-Valentine leukocidin), hlg (gamma-toxin), alpha-toxin and icaappeared to be unrelated to the underlying clonal structure suggestingconsiderable horizontal transfer of these genes.

In contrary, other virulence genes such as fibronectin binding protein B(fnbB), beta-hemolysin (hlb), collagen binding protein (cna), TSST-1(tst) and methicillin resistance gene (mecA) are strongly associatedwith specific lineages (Booth et al., 2001 Infect Immun. 69(1):345-52).Similarly, Peacock et al. 2002 (Infect Immun. 70(9):4987-96) showed thatthe distributions of the enterotoxins, tst, the exfolatins (eta andetb), beta- and delta-toxins, the sdr genes (sdrD, sdrE and bbp), cna,ebpS and efb within the population are all highly significantly relatedto MLST-derived clonal complexes.

MLST data provide no evidence that strains responsible for nosocomialdisease represent a distinct subpopulation from strains causingcommunity-acquired disease or strains recovered from asymptomaticcarriers (Feil et al., 2003 J. Bacteriol. 185(11):3307-16).

Preferred immunogenic compositions of the invention are effectiveagainst staphylococci from different clonal lineages.

In an embodiment, the immunogenic composition comprises 1, 2, 3, 4,preferably at least 1 protein that is expressed in most isolates ofstaphylococci. Examples of such proteins include alpha-hemolysin (hla),fibronectin-binding protein A (fnbA) and clumping factor A (clfA),coagulase, spa, map, pvl (Panton-Valentine leukocidin), hlg(gamma-toxin), ica, immunodominant ABC transporter, RAP, autolysin (Ruppet al 2001, J. Infect. Dis. 183; 1038), laminin receptors, SitC,IsaA/PisA, SPOIIIE ( ) SsaA, EbpS, SasF (Roche et al 2003, Microbiology149; 643), EFB(FIB), SBI, ClfB, IsdA, IsdB, FnbB, Npase, EBP, Bone sialobinding protein II, IsaB/PisB (Lorenz et al FEMS Immuno. Med. Microb.2000, 29; 145), SasH (Roche et at 2003, Microbiology 149; 643), MRPI,SasD (Roche et al 2003, Microbiology 149; 643), SasH (Roche et al 2003,Microbiology 149; 643), aureolysin precursor (AUR)/Sepp1 and novelautolysin.

In an alternative embodiment, 2 or more proteins which are expressed indifferent sets of clonal strains are included in the immunogeniccomposition of the invention. Preferably the combination of antigenswill allow an immune response to be generated that is effective againstmultiple clonal strains, most preferably against all clonal stains.Preferred combinations include FnbB and betahemolysin, FnbB and Cna,FnbB and TSST-1, FnbB and mecA, FnbB and SdrD, FnbB and SdrF, FnbB andEbpS, FnbB and Efb, beta-haemolysin and Cna, beta-haemolysin and TSST-1,beta-haemolysin and mecA, beta-haemolysin and SdrD, beta-haemolysin andSdrF, beta-haemolysin and EbpS, beta-haemolysin and Efb, Cna and TSST-1,Cna and mecA, Cna and SdrD, Cna and SdrF, Cna and EbpS, Cna and Efb,TSST-1 and mecA, TSST-1 and SdrD, TSST-1 and SdrF, TSST-1 and EbpS,TssT-1 and Efb, MecA and SdrD, MecA and SdrF, MecA and EbpS, MecA andEfb, SdrD and SdrF, SdrD and EbpS, SdeD and Efb, SdrF and EbpS, SdrF andEfb, and, EbpS and Efb.

The preferred combinations described above may be combined withadditional components described above.

Protection Against S. aureus and S. epidermidis

In a preferred embodiment of the invention the immunogenic compositionprovides an effective immune response against more than one strain ofstaphylococci, preferably against strains from both S. aureus and S.epidermidis. More preferably, a protective immune response is generatedagainst type 5 and 8 serotypes of S. aureus. More preferably, aprotective immune response is generated against serotypes I, II and IIIof S. epidermidis.

One use of the immunogenic composition of the invention is to preventnosocomial infections, for instance in elective surgery patients, byinoculating prior to hospital treatment. At this stage, it is difficultto accurately predict which staphylococcal strains the patient will beexposed to. It is therefore advantageous to inoculate with a vaccinethat is capable of generating an effective immune response againstvarious strains of staphylococci.

An effective immune response is defined as an immune response that givessignificant protection in a mouse challenge model or opsonophagocytosisassay as described in the examples. Significant protection in a mousechallenge model, for instance that of example 5, is defined as anincrease in the LD50 in comparison with carrier inoculated mice of atleast 10%, 20%, 50%, 100% or 200%. Significant protection in a cottonrat challenge model, for instance that of example 8, is defined as adecrease in the mean observed LogCFU/nose of at least 10%, 20%, 50%, 70%or 90%. The presence of opsonising antibodies is known to correlate withprotection, therefore significant protection is indicated by a decreasein the bacterial count of at least 10%, 20%, 50%, 70% or 90% in anopsonophagocytosis assay, for instance that of example 7.

Several of the proteins including immunodominant ABC transporter, RNAIII activating protein, Laminin receptors, SitC, IsaA/PisA, SsaA,EbhA/EbhB, EbpS and Aap are well conserved between S. aureus and S.epidermidis and example 8 shows that IsaA, ClfA, IsdB, SdrG, HarA, FnbpAand Sbi can generate a cross-reactive immune response (for examplecrossreactive between at least one S. aureus and at least one S.epidermidis strain). PIA is also well conserved between S. aureus and S.epidermidis.

Therefore in a preferred embodiment, the immunogenic composition of theinvention will comprise PIA and type 5 and 8 polysaccharides and willfurther comprise one, two, three or four of the above proteins.

Vaccines

In a preferred embodiment, the immunogenic composition of the inventionis mixed with a pharmaceutically acceptable excipient, more preferablywith an adjuvant to form a vaccine.

The vaccines of the present invention are preferably adjuvanted.Suitable adjuvants include an aluminum salt such as aluminum hydroxidegel (alum) or aluminium phosphate, but may also be a salt of calcium,magnesium, iron or zinc, or may be an insoluble suspension of acylatedtyrosine, or acylated sugars, cationically or anionically derivatizedpolysaccharides, or polyphosphazenes.

It is preferred that the adjuvant be selected to be a preferentialinducer of either a TH1 or a TH2 type of response. High levels ofTh1-type cytokines tend to favor the induction of cell mediated immuneresponses to a given antigen, whilst high levels of Th2-type cytokinestend to favour the induction of humoral immune responses to the antigen.

It is important to remember that the distinction of Th1 and Th2-typeimmune response is not absolute. In reality an individual will supportan immune response which is described as being predominantly Th1 orpredominantly Th2. However, it is often convenient to consider thefamilies of cytokines in terms of that described in murine CD4 +ve Tcell clones by Mosmann and Coffman (Mosmann, T. R. and Coffman, R. L.(1989) TH1 and TH2 cells: different patterns of lymphokine secretionlead to different functional properties. Annual Review of Immunology, 7,p 145-173). Traditionally, Th1-type responses are associated with theproduction of the INF-γ and IL-2 cytokines by T-lymphocytes. Othercytokines often directly associated with the induction of Th1-typeimmune responses are not produced by T-cells, such as IL-12. Incontrast, Th2-type responses are associated with the secretion of Il-4,IL-5, IL-6, IL-10. Suitable adjuvant systems which promote apredominantly Th1 response include: Monophosphoryl lipid A or aderivative thereof, particularly 3-de-O-acylated monophosphoryl lipid A(3D-MPL) (for its preparation see GB 2220211 A); and a combination ofmonophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipidA, together with either an aluminium salt (for instance aluminiumphosphate or aluminium hydroxide) or an oil-in-water emulsion. In suchcombinations, antigen and 3D-MPL are contained in the same particulatestructures, allowing for more efficient delivery of antigenic andimmunostimulatory signals. Studies have shown that 3D-MPL is able tofurther enhance the immunogenicity of an alum-adsorbed antigen [Thoelenet al. Vaccine (1998) 16:708-14; EP 689454-B1].

An enhanced system involves the combination of a monophosphoryl lipid Aand a saponin derivative, particularly the combination of QS21 and3D-MPL as disclosed in WO 94/00153, or a less reactogenic compositionwhere the QS21 is quenched with cholesterol as disclosed in WO 96/33739.A particularly potent adjuvant formulation involving QS21, 3D-MPL andtocopherol in an oil in water emulsion is described in WO 95/17210, andis a preferred formulation. Preferably the vaccine additionallycomprises a saponin, more preferably QS21. The formulation may alsocomprise an oil in water emulsion and tocopherol (WO 95/17210). Thepresent invention also provides a method for producing a vaccineformulation comprising mixing a protein of the present inventiontogether with a pharmaceutically acceptable excipient, such as 3D-MPL.Unmethylated CpG containing oligonucleotides (WO 96/02555) are alsopreferential inducers of a TH1 response and are suitable for use in thepresent invention.

Preferred compositions of the invention are those forming a liposomestructure. Compositions where the sterol/immunologically active saponinfraction forms an ISCOM structure also form an aspect of the invention.

The ratio of QS21: sterol will typically be in the order of 1:100 to 1:1weight to weight. Preferably excess sterol is present, the ratio ofQS21: sterol being at least 1:2 w/w. Typically for human administrationQS21 and sterol will be present in a vaccine in the range of about 1 μgto about 100 μg, preferably about 10 μg to about 50 μg per dose.

The liposomes preferably contain a neutral lipid, for examplephosphatidylcholine, which is preferably non-crystalline at roomtemperature, for example eggyolk phosphatidylcholine, dioleoylphosphatidylcholine or dilauryl phosphatidylcholine. The liposomes mayalso contain a charged lipid which increases the stability of thelipsome-QS21 structure for liposomes composed of saturated lipids. Inthese cases the amount of charged lipid is preferably 1-20% w/w, mostpreferably 5-10%. The ratio of sterol to phospholipid is 1-50%(mol/mol), most preferably 20-25%.

Preferably the compositions of the invention contain MPL (3-deacylatedmono-phosphoryl lipid A, also known as 3D-MPL). 3D-MPL is known from GB2 220 211 (Ribi) as a mixture of 3 types of De-O-acylated monophosphoryllipid A with 4, 5 or 6 acylated chains and is manufactured by RibiImmunochem, Montana. A preferred form is disclosed in InternationalPatent Application 92/116556.

Suitable compositions of the invention are those wherein liposomes areinitially prepared without MPL, and MPL is then added, preferably as 100nm particles. The MPL is therefore not contained within the vesiclemembrane (known as MPL out). Compositions where the MPL is containedwithin the vesicle membrane (known as MPL in) also form an aspect of theinvention. The antigen can be contained within the vesicle membrane orcontained outside the vesicle membrane. Preferably soluble antigens areoutside and hydrophobic or lipidated antigens are either containedinside or outside the membrane.

The vaccine preparations of the present invention may be used to protector treat a mammal susceptible to infection, by means of administeringsaid vaccine via systemic or mucosal route. These administrations mayinclude injection via the intramuscular, intraperitoneal, intradermal orsubcutaneous routes; or via mucosal administration to theoral/alimentary, respiratory, genitourinary tracts. Intranasaladministration of vaccines for the treatment of pneumonia or otitismedia is preferred (as nasopharyngeal carriage of pneumococci can bemore effectively prevented, thus attenuating infection at its earlieststage). Although the vaccine of the invention may be administered as asingle dose, components thereof may also be co-administered together atthe same time or at different times (for instance pneumococcalpolysaccharides could be administered separately, at the same time or1-2 weeks after the administration of any bacterial protein component ofthe vaccine for optimal coordination of the immune responses withrespect to each other). For co-administration, the optional Th1 adjuvantmay be present in any or all of the different administrations, howeverit is preferred if it is present in combination with the bacterialprotein component of the vaccine. In addition to a single route ofadministration, 2 different routes of administration may be used. Forexample, polysaccharides may be administered IM (or ID) and bacterialproteins may be administered IN (or ID). In addition, the vaccines ofthe invention may be administered IM for priming doses and IN forbooster doses.

The amount of conjugate antigen in each vaccine dose is selected as anamount which induces an immunoprotective response without significant,adverse side effects in typical vaccines. Such amount will varydepending upon which specific immunogen is employed and how it ispresented. Generally, it is expected that each dose will comprise0.1-100 μg of polysaccharide, preferably 0.1-50 μg for polysaccharideconjugates, preferably 0.1-10 μg, more preferably 1-10 μg, of which 1 to5 μg is a more preferable range. However, for serotype 6B, the preferreddosage will comprise 3-10 μg of polysaccharide.

The content of protein antigens in the vaccine will typically be in therange 1-100 μg, preferably 5-50 μg, most typically in the range 5-25 μg.Following an initial vaccination, subjects may receive one or severalbooster immunizations adequately spaced.

Vaccine preparation is generally described in Vaccine Design (“Thesubunit and adjuvant approach” (eds Powell M. F. & Newman M. J.) (1995)Plenum Press New York). Encapsulation within liposomes is described byFullerton, U.S. Pat. No. 4,235,877.

The vaccines of the present invention may be stored in solution orlyophilized. Preferably the solution is lyophilized in the presence of asugar such as sucrose, trehalose or lactose. It is still furtherpreferable that they are lyophilized and extemporaneously reconstitutedprior to use. Lyophilizing may result in a more stable composition(vaccine) and may possibly lead to higher antibody titers in thepresence of 3D-MPL and in the absence of an aluminium based adjuvant.

Antibodies and Passive Immunisation

Another aspect of the invention is a method of preparing an immuneglobulin for use in prevention or treatment of staphylococcal infectioncomprising the steps of immunising a recipient with the vaccine of theinvention and isolating immune globulin from the recipient. An immuneglobulin prepared by this method is a further aspect of the invention. Apharmaceutical composition comprising the immune globulin of theinvention and a pharmaceutically acceptable carrier is a further aspectof the invention which could be used in the manufacture of a medicamentfor the treatment or prevention of staphylococcal disease. A method fortreatment or prevention of staphylococcal infection comprising a step ofadministering to a patient an effective amount of the pharmaceuticalpreparation of the invention is a further aspect of the invention.

Inocula for polyclonal antibody production are typically prepared bydispersing the antigenic composition in a physiologically tolerablediluent such as saline or other adjuvants suitable for human use to forman aqueous composition. An immunostimulatory amount of inoculum isadministered to a mammal and the inoculated mammal is then maintainedfor a time sufficient for the antigenic composition to induce protectiveantibodies.

The antibodies can be isolated to the extent desired by well knowntechniques such as affinity chromatography (Harlow and Lane Antibodies;a laboratory manual 1988).

Antibodies can include antiserum preparations from a variety of commonlyused animals e.g. goats, primates, donkeys, swine, horses, guinea pigs,rats or man. The animals are bled and serum recovered.

An immune globulin produced in accordance with the present invention caninclude whole antibodies, antibody fragments or subfragments. Antibodiescan be whole immunoglobulins of any class e.g. IgG, IgM, IgA, IgD orIgE, chimeric antibodies or hybrid antibodies with dual specificity totwo or more antigens of the invention. They may also be fragments e.g.F(ab′)2, Fab′, Fab, Fv and the like including hybrid fragments. Animmune globulin also includes natural, synthetic or geneticallyengineered proteins that act like an antibody by binding to specificantigens to form a complex.

A vaccine of the present invention can be administered to a recipientwho then acts as a source of immune globulin, produced in response tochallenge from the specific vaccine. A subject thus treated would donateplasma from which hyperimmune globulin would be obtained viaconventional plasma fractionation methodology. The hyperimmune globulinwould be administered to another subject in order to impart resistanceagainst or treat staphylococcal infection. Hyperimmune globulins of theinvention are particularly useful for treatment or prevention ofstaphylococcal disease in infants, immune compromised individuals orwhere treatment is required and there is no time for the individual toproduce antibodies in response to vaccination.

An additional aspect of the invention is a pharmaceutical compositioncomprising two of more monoclonal antibodies (or fragments thereof;preferably human or humanised) reactive against at least twoconstituents of the immunogenic composition of the invention, whichcould be used to treat or prevent infection by Gram positive bacteria,preferably staphylococci, more preferably S. aureus or S. epidermidis.

Such pharmaceutical compositions comprise monoclonal antibodies that canbe whole immunoglobulins of any class e.g. IgG, IgM, IgA, IgD or IgE,chimeric antibodies or hybrid antibodies with specificity to two or moreantigens of the invention. They may also be fragments e.g. F(ab′)2,Fab′, Fab, Fv and the like including hybrid fragments.

Methods of making monoclonal antibodies are well known in the art andcan include the fusion of splenocytes with myeloma cells (Kohler andMilstein 1975 Nature 256; 495; Antibodies—a laboratory manual Harlow andLane 1988). Alternatively, monoclonal Fv fragments can be obtained byscreening a suitable phage display library (Vaughan T J et al 1998Nature Biotechnology 16; 535). Monoclonal antibodies may be humanised orpart humanised by known methods.

Methods

The invention also encompasses method of making the immunogeniccompositions and vaccines of the invention.

A preferred process of the invention, is a method to make a vaccinecomprising the steps of mixing antigens to make the immunogeniccomposition of the invention and adding a pharmaceutically acceptableexcipient.

Methods of Treatment

The invention also encompasses method of treatment or staphylococcalinfection, particularly hospital acquired nosocomial infections.

This immunogenic composition or vaccine of the invention is particularlyadvantageous to use in cases of elective surgery. Such patients willknow the date of surgery in advance and could be inoculated in advance.Since it is not know whether the patient will be exposed to S. aureus orS. epidermidis infection, it is preferred to inoculate with a vaccine ofthe invention that protects against both, as described above. Preferablyadults over 16 awaiting elective surgery are treated with theimmunogenic compositions and vaccines of the invention.

It is also advantageous to inoculate health care workers with thevaccine of the invention.

The vaccine preparations of the present invention may be used to protector treat a mammal susceptible to infection, by means of administeringsaid vaccine via systemic or mucosal route. These administrations mayinclude injection via the intramuscular, intraperitoneal, intradermal orsubcutaneous routes; or via mucosal administration to theoral/alimentary, respiratory, genitourinary tracts.

The amount of antigen in each vaccine dose is selected as an amountwhich induces an immunoprotective response without significant, adverseside effects in typical vaccines. Such amount will vary depending uponwhich specific immunogen is employed and how it is presented. Theprotein content of the vaccine will typically be in the range 1-100 μg,preferably 5-50 μg, most typically in the range 10-25 μg. Generally, itis expected that each dose will comprise 0.1-100 μg of polysaccharidewhere present, preferably 0.1-50 μg, preferably 0.1-10 μg, of which 1 to5 μg is the most preferable range. An optimal amount for a particularvaccine can be ascertained by standard studies involving observation ofappropriate immune responses in subjects. Following an initialvaccination, subjects may receive one or several booster immunisationsadequately spaced.

Although the vaccines of the present invention may be administered byany route, administration of the described vaccines into the skin (ID)forms one embodiment of the present invention. Human skin comprises anouter “horny” cuticle, called the stratum corneum, which overlays theepidermis. Underneath this epidermis is a layer called the dermis, whichin turn overlays the subcutaneous tissue. Researchers have shown thatinjection of a vaccine into the skin, and in particular the dermis,stimulates an immune response, which may also be associated with anumber of additional advantages. Intradermal vaccination with thevaccines described herein forms a preferred feature of the presentinvention.

The conventional technique of intradermal injection, the “mantouxprocedure”, comprises steps of cleaning the skin, and then stretchingwith one hand, and with the bevel of a narrow gauge needle (26-31 gauge)facing upwards the needle is inserted at an angle of between 10-15°.Once the bevel of the needle is inserted, the barrel of the needle islowered and further advanced whilst providing a slight pressure toelevate it under the skin. The liquid is then injected very slowlythereby forming a bleb or bump on the skin surface, followed by slowwithdrawal of the needle.

More recently, devices that are specifically designed to administerliquid agents into or across the skin have been described, for examplethe devices described in WO 99/34850 and EP 1092444, also the jetinjection devices described for example in WO 01/13977; U.S. Pat. No.5,480,381, U.S. Pat. No. 5,599,302, U.S. Pat. No. 5,334,144, U.S. Pat.No. 5,993,412, U.S. Pat. No. 5,649,912, U.S. Pat. No. 5,569,189, U.S.Pat. No. 5,704,911, U.S. Pat. No. 5,383,851, U.S. Pat. No. 5,893,397,U.S. Pat. No. 5,466,220, U.S. Pat. No. 5,339,163, U.S. Pat. No.5,312,335, U.S. Pat. No. 5,503,627, U.S. Pat. No. 5,064,413, U.S. Pat.No. 5,520,639, U.S. Pat. No. 4,596,556, U.S. Pat. No. 4,790,824, U.S.Pat. No. 4,941,880, U.S. Pat. No. 4,940,460, WO 97/37705 and WO97/13537. Alternative methods of intradermal administration of thevaccine preparations may include conventional syringes and needles, ordevices designed for ballistic delivery of solid vaccines (WO 99/27961),or transdermal patches (WO 97/48440; WO 98/28037); or applied to thesurface of the skin (transdermal or transcutaneous delivery WO 98/20734;WO 98/28037).

When the vaccines of the present invention are to be administered to theskin, or more specifically into the dermis, the vaccine is in a lowliquid volume, particularly a volume of between about 0.05 ml and 0.2ml.

The content of antigens in the skin or intradermal vaccines of thepresent invention may be similar to conventional doses as found inintramuscular vaccines (see above). However, it is a feature of skin orintradermal vaccines that the formulations may be “low dose”.Accordingly the protein antigens in “low dose” vaccines are preferablypresent in as little as 0.1 to 10 μg, preferably 0.1 to 5 μg per dose;and the polysaccharide (preferably conjugated) antigens may be presentin the range of 0.01-1 μg, and preferably between 0.01 to 0.5 μg ofpolysaccharide per dose.

As used herein, the term “intradermal delivery” means delivery of thevaccine to the region of the dermis in the skin. However, the vaccinewill not necessarily be located exclusively in the dermis. The dermis isthe layer in the skin located between about 1.0 and about 2.0 mm fromthe surface in human skin, but there is a certain amount of variationbetween individuals and in different parts of the body. In general, itcan be expected to reach the dermis by going 1.5 mm below the surface ofthe skin. The dermis is located between the stratum corneum and theepidermis at the surface and the subcutaneous layer below. Depending onthe mode of delivery, the vaccine may ultimately be located solely orprimarily within the dermis, or it may ultimately be distributed withinthe epidermis and the dermis.

A preferred embodiment of the invention is a method of preventing ortreating staphylococcal infection or disease comprising the step ofadministering the immunogenic composition or vaccine of the invention toa patient in need thereof.

In a preferred embodiment, the patient is awaiting elective surgery.

A further preferred embodiment of the invention is a use of theimmunogenic composition of the invention in the manufacture of a vaccinefor treatment or prevention of staphylococcal infection or disease,preferably post-surgery staphylococcal infection.

The term ‘staphylococcal infection’ encompasses infection caused by S.aureus and/or S. epidermidis and other staphylococcal strains capable ofcausing infection in a mammalina, preferably human host.

The terms “comprising”, “comprise” and “comprises” herein are intendedby the inventors to be optionally substitutable with the terms“consisting of”, “consist of” and “consists of”, respectively, in everyinstance.

All references or patent applications cited within this patentspecification are incorporated by reference herein.

In order that this invention may be better understood, the followingexamples are set forth. These examples are for purposes of illustrationonly, and are not to be construed as limiting the scope of the inventionin any manner.

EXAMPLES Example 1 Construction of Plasmid to Express Recombinantproteins A: Cloning.

Appropriate restriction sites engineered into oligonucleotides specificfor the staphylococcal gene permitted directional cloning of the PCRproduct into the E. coli expression plasmid pET24d or pQE-30 such that aprotein could be expressed as a fusion protein containing a (His)6affinity chromatography tag at the N- or C-terminus.

The primers used were:

Alpha toxin- 5′-CGCGGATCCGCAGATTCTGATATTAATATTAAAAC-3′ and5′CCCAAGCTTTTAATTTGTCATTTCTTCTTTTTC-3′ EbpS-5′-CGCGGATCCGCTGGGTCTAATAATTTTAAAGATG-3′ and5′CCCAAGCTTTTATGGAATAACGATTTGTTG-3′ ClfA-5′-CGCGGATCCAGTGAAAATAGTGTTACGCAATC-3′ and5′CCCAAGCTTTTACTCTGGAATTGGTTCAATTTC-3′ FnbpA-5′-CGCGGATCCACACAAACAACTGCAACTAACG-3′ and5′CCCAAGCTTTTATGCTTTGTGATTCTTTTTCAAAC3′ Sbi-5′-CGCGGATCCAACACGCAACAAACTTC-3′ and5′GGAACTGCAGTTATTTCCAGAATGATAATAAATTAC-3′ SdrC-5′-CGCGGATCCGCAGAACATACGAATGGAG-3′ and5′CCCAAGCTTTTATGTTTCTTCTTCGTAGTAGC-3′ SdrG-5′-CGCGGATCCGAGGAGAATTCAGTACAAG-3′ and5′CCCAAGCTTTTATTCGTCATCATAGTATCCG-3′ Ebh-5′-AAAAGTACTCACCACCACCACCACC-3′ and 5′AAAAGTACTCACTTGATTCATCGCTICAG-3′Aaa- 5′-GCGCGCCATGGCACAAGCTTCTACACAACATAC-3′ and5′GCGCGCTCGAGATGGATGAATGCATAGCTAGA-3′ IsaA-5′-GCATCCATGGCACCATCACCATCACCACGAAGTAAACGTTGAT CAAGC-3′ and5′-AGCACTCGAGTTAGAATCCCCAAGCACCTAAACC-3′ HarA-5′-GCACCCATGGCAGAAAATACAAATACTTC-3′ and5′TTTTCTCGAGCATTTTAGATTGACTAAGTTG-3′ Autolysin glucosaminidase-5′-CAAGTCCCATGGCTGAGACGACACAAGATCAAC-3′ and5′-CAGTCTCGAGTTTTACAGCTGTTTTTGGTTG-3′ Autolysin amidase-5′-AGCTCATATGGCTTATACTGTTACTAAACC-3′ and5′GCGCCTCGAGTTTATATTGTGGGATGTCG-3′ IsdA-5′-CAAGTCCCATGGCAACAGAAGCTACGAACGCAAC-3′ and5′ACCAGTCTCGAGTAATTCTTTAGCTTTAGAGCTTG-3′ IsdB-5′-TATTCTCGAGGCTTTGAGTGTGTCCATCATTTG-3′ and5′GAAGCCATGGCAGCAGCTGAAGAAACAGGTGG-3′ MRPII-5′-GATTACACCATGGTTAAACCTCAAGCGAAA-3′ and5′AGGTGTCTCGAGTGCGATTGTAGCTTCATT-3′

The PCR products were first introduced into the pGEM-T cloning vector(Novagen) using Top10 bacterial cells, according to the manufacturer'sinstructions. This intermediate construct was made to facilitate furthercloning into an expression vector. Transformants containing the DNAinsert were selected by restriction enzyme analysis. Followingdigestion, a ˜20 μl aliquot of the reaction was analyzed by agarose gelelectrophoresis (0.8% agarose in a Tris-acetate-EDTA (TAE) buffer). DNAfragments were visualized by UV illumination after gel electrophoresisand ethidium bromide staining. A DNA molecular size standard (1 Kbladder, Life Technologies) was electrophoresed in parallel with the testsamples and was used to estimate the size of the DNA fragments. Plasmidpurified from selected transformants for each cloning was thensequentially digested to completion with appropriate restriction enzymesas recommended by the manufacturer (Life Technologies). The digested DNAfragment was then purified using silica gel-based spin columns prior toligation with the pET24d or pQE-30 plasmid. Cloning of Ebh (H2fragment), AaA, IsdA, IsdB, HarA, Atl-amidase, Atl-glucosamine, MRP,IsaA was carried out using the pET24d plasmid and cloning of ClfA, SdrC,SdrE, FnbpA, SdrG/Fbe, alpha toxin and Sbi were carried out using thepQE-30 plasmid.

B: Production of Expression Vector.

To prepare the expression plasmid pET24d or pQE-30 for ligation, it wassimilarly digested to completion with appropriate restriction enzymes.An approximately 5-fold molar excess of the digested fragments to theprepared vector was used to program the ligation reaction. A standard˜20 μl ligation reaction (˜16° C., ˜16 hours), using methods well knownin the art, was performed using T4 DNA ligase (˜2.0 units/reaction, LifeTechnologies). An aliquot of the ligation (˜5 μl) was used to transformM15(pREP4) or BT21::DE3 electro-competent cells according to methodswell known in the art. Following a ˜2-3 hour outgrowth period at 37° C.in ˜1.0 ml of LB broth, transformed cells were plated on LB agar platescontaining ampicillin (100 μg/ml) and/or kanamycin (30 μg/ml).Antibiotics were included in the selection. Plates were incubatedovernight at 37° C. for ˜16 hours. Individual ApR/KanR colonies werepicked with sterile toothpicks and used to “patch” inoculate fresh LBApR/KanR plates as well as a ˜1.0 ml LB Ap/Kan broth culture. Both thepatch plates and the broth culture were incubated overnight at 37° C. ineither a standard incubator (plates) or a shaking water bath. A wholecell-based PCR analysis was employed to verify that transformantscontained the DNA insert. Here, the ˜1.0 ml overnight LB Ap/Kan brothculture was transferred to a 1.5 ml polypropylene tube and the cellscollected by centrifugation in a Beckmann microcentrifuge (˜3 min., roomtemperature, ˜12,000×g). The cell pellet was suspended in ˜200 μl ofsterile water and a ˜10 μl aliquot used to program a ˜50 μl final volumePCR reaction containing both forward and reverse amplification primers.The initial 95° C. denaturation step was increased to 3 minutes toensure thermal disruption of the bacterial cells and liberation ofplasmid DNA. An ABI Model 9700 thermal cycler and a 32 cycle, three-stepthermal amplification profile, i.e. 95° C., 45 sec; 55-58° C., 45 sec,72° C., 1 min., were used to amplify the BASB203 fragment from the lysedtransformant samples. Following thermal amplification, a ˜20 μl aliquotof the reaction was analyzed by agarose gel electrophoresis (0.8%agarose in a Tris-acetate-EDTA (TAE) buffer). DNA fragments werevisualised by UV illumination after gel electrophoresis and ethidiumbromide staining. A DNA molecular size standard (1 Kb ladder, LifeTechnologies) was electrophoresed in parallel with the test samples andwas used to estimate the size of the PCR products. Transformants thatproduced the expected size PCR product were identified as strainscontaining a protein expression construct. Expression plasmid containingstrains were then analyzed for the inducible expression of recombinantprotein.

C: Expression Analysis of PCR-Positive Transformants.

An aliquot of the overnight seed culture (˜1.0 ml) was inoculated into a125 ml erlenmeyer flask containing ˜25 ml of LB Ap/Kan broth and wasgrown at 37° C. with shaking (−250 rpm) until the culture turbidityreached O.D.600 of ˜0.5, i.e. mid-log phase (usually about 1.5-2.0hours). At this time approximately half of the culture (˜12.5 ml) wastransferred to a second 125 ml flask and expression of recombinantprotein induced by the addition of IPTG (1.0 M stock prepared in sterilewater, Sigma) to a final concentration of 1.0 mM. Incubation of both theIPTG-induced and non-induced cultures continued for an additional ˜4hours at 37° C. with shaking. Samples (˜1.0 ml) of both induced andnon-induced cultures were removed after the induction period and thecells collected by centrifugation in a microcentrifuge at roomtemperature for ˜3 minutes. Individual cell pellets were suspended in˜50 μl of sterile water, then mixed with an equal volume of 2× LaemelliSDS-PAGE sample buffer containing 2-mercaptoethanol, and placed inboiling water bath for ˜3 min to denature protein. Equal volumes (˜15μl) of both the crude IPTG-induced and the non-induced cell lysates wereloaded onto duplicate 12% Tris/glycine polyacrylamide gel (1 mm thickMini-gels, Novex). The induced and non-induced lysate samples wereelectrophoresed together with prestained molecular weight markers(SeeBlue, Novex) under conventional conditions using a standardSDS/Tris/glycine running buffer (BioRad). Following electrophoresis, onegel was stained with commassie brilliant blue R250 (BioRad) and thendestained to visualize novel IPTG-inducible protein(s). The second gelwas electroblotted onto a PVDF membrane (0.45 micron pore size, Novex)for ˜2 hrs at 4° C. using a BioRad Mini-Protean II blotting apparatusand Towbin's methanol (20%) transfer buffer. Blocking of the membraneand antibody incubations were performed according to methods well knownin the art. A monoclonal anti-RGS (His)3 antibody, followed by a secondrabbit anti-mouse antibody conjugated to HRP (QiaGen), were used toconfirm the expression and identity of the recombinant protein.Visualization of the anti-His antibody reactive pattern was achievedusing either an ABT insoluble substrate or using Hyperfilm with theAmersham ECL chemiluminescence system.

Example 2 Production of Recombinant Protein Bacterial Strain

A recombinant expression strain of E. coli M15(pREP4) containing aplasmid (pQE30) or BL21::DE3 containing plasmid pET24d encodingstaphylococcal protein was used to produce cell mass for purification ofrecombinant protein.

Media

The fermentation medium used for the production of recombinant proteinconsisted of 2×YT broth (Difco) containing 100 μg/ml Ap and/or 30 μg/mlKm. Antifoam was added to medium for the fermentor at 0.25 ml/L(Antifoam 204, Sigma). To induce expression of the recombinant protein,IPTG (Isopropyl β-D-Thiogalactopyranoside) was added to the fermentor (1mM, final).

Production of Recombinant Proteins Under Native Conditions

IPTG was added at a final concentration of 1 mM and the culture wasgrown for 4 additional hours. The culture was then centrifuged at 6,000rpm for 10 minutes and the pellet was resuspended in phosphate buffer(50 mM K2HPO4, KH2PO4 pH 7) including a protease inhibitor cocktail.This sample was subjected to French pressure lysis using 1500 barpressure (2 runs). After centrifugation for 30 minutes at 15,000 rpm,the supernatant was reserved for further purification and NaCl was addedto 0.5M. The sample was then loaded on a Ni-NTA resin (XK 16 columnPharmacia, Ni-NTA resin Qiagen) conditioned in 50 mM K2HPO4, KH2PO4 pH7. After loading the sample, the column was washed with Buffer A (0.2MNaH2PO4 pH7, 0.3M NaCl, 10% glycerol). To elute bound protein, a stepgradient was used where different proportions of buffer B (0.2M NaH2PO4pH7, 0.3M NaCl, 10% glycerol and 200 mM imidazole) were added to bufferA. The proportion of buffer B was gradually increased from 10% to 100%.After purification, eluted fraction containing the protein were pooled,concentrated and dialysed against 0.002M KH2PO4/K2HPO4 pH7, 0.15M NaCl.

This method was used to purify ClfA, SdrG, IsdA, IsaB, HarA,Atl-glucosamine and alpha toxin.

Under Denaturing Conditions

IPTG was added at a final concentration of 1 mM and the culture wasgrown for 4 additional hours. The culture was then centrifuged at 6,000rpm for 10 minutes and the pellet was resuspended in phosphate buffer(50 mM K2HPO4, KH2PO4 pH 7) including a protease inhibitor cocktail.This sample was subjected to French pressure lysis using 1500 barpressure (2 runs). After centrifugation for 30 minutes at 15,000 rpm,the pellet was washed with phosphate buffer including 1M urea. Thesample was centrifuged for 30 mins at 15000 rpm and the pellet wasresuspended in 8M urea, 0.1M NaH2PO4, 0.5M NaCl, 0.01 M Tris-Hcl pH8 andkept overnight at room temperature. The sample was centrifuged fro 20minutes at 15000 rpm and the supernatant was collected for furtherpurification. The sample was then loaded on a Ni-NTA resin (XK 16 columnPharmacia, Ni-NTA resin Qiagen) conditioned in 8M urea, 0.1M NaH2PO4,0.5M NaCl, 0.01M Tris-Hcl pH8. After passage of the flowthrough, thecolumn was washed successively with buffer A (8M Urea, 0.1MNaH2PO4, 0.5MNaCl, 0.01M Tris, pH 8.0), buffer C (8M Urea, 0.1MNaH2PO4, 0.5M NaCl,0.01M Tris, pH 6.3), buffer D (8M Urea, 0.1MNaH2PO4, 0.5M NaCl, 0.01MTris, pH 5.9) and buffer E (8M Urea, 0.1MNaH2PO4, 0.5M NaCl, 0.01M Tris,pH 4.5). The recombinant protein was eluted from the column duringwashes with buffer D and E. The denatured, recombinant protein could besolubilized in a solution devoid of urea. For this purpose, denaturedprotein contained in 8M urea was successively dialyzed against 4M urea,0.1MNa2PO_(4, 0.01)M Tris-HCl, pH7.1, 2M urea, 0.1 M NaH2PO4, 0.01MTris-HCl, pH 7.1, 0.5M arginine and 0.002M KH2PO4/K2HPO4 pH7.1, 0.15MNaCl, 0.5M arginine.

This method was used to purify Ebh (H2 fragment), AaA, SdrC, FnbpA, Sbi,Atl-amidase and IsaA.

The purified proteins were analysed by SDS-PAGE. The results for oneprotein purified under native conditions (alpha toxin) and one proteinpurified under denaturing conditions (SdrC) are shown in FIGS. 3 and 4.

Example 3 Preparation of Polysaccharides

PNAG is prepared as described in Joyce et al 2003, Carbohydrate Research338; 903-922.

Type 5 and type 8 polysaccharides are extracted from S. aureus asdescribed in Infection and Immunity (1990) 58(7); 2367.

LTA is extracted from staphylococci as described in Fischer W, et alEur. J. Biochem. (1983) 133; 523 or as described in Morath et al J. Exp.Med. 2001; 193; 393-397.

Activation and Coupling Chemistry:

Native polysaccharide is dissolved in NaCl 2M or in water. The optimalpolysaccharide concentration is evaluated for all the serotypes and isbetween 2 mg/ml and 5 mg/ml.

From a 100 mg/ml stock solution in acetonitrile, CDAP(CDAP/PS ratio:0.75mg/mg PS) is added to the polysaccharide solution.1.5 minute later, 0.2Mtriethylamine is added to obtain the specific activation pH (pH8.5-10.0). The activation of the polysaccharide is performed at this pHduring 2 minutes at 25° C. The carrier protein is added to the activatedpolysaccharide in an amount sufficient to give a 1/1 molar ratio and thecoupling reaction is performed at the specific pH for 1 hour.

Then, the reaction is quenched with glycine for 30 minutes at 25° C. andovernight at 4° C.

The conjugates are purified by gel filtration using a Sephacryl 500HRgel filtration column equilibrated with 0.2M NaCl.

The carbohydrate and protein contents of the eluted fractions aredetermined. The conjugates are pooled and sterile filtered on a 0.22 μmsterilizing membrane. The PS/Protein ratios in the conjugatepreparations are determined.

Characterisation:

Each conjugate is characterised for protein and polysaccharide content.

The polysaccharide content is measured by the Resorcinol test and theprotein content by the Lowry test. The final PS/PD ratio(w/w) isdetermined by the ratio of the concentrations.

Residual DMAP Content (ng/μg PS):

The activation of the polysaccharide with CDAP introduces a cyanategroup in the polysaccharide and DMAP (4-dimethylamino-pyridin) isliberated. The residual DMAP content is determined by a specific assaydeveloped and validated at GSK.

Free Polysaccharide Content (%):

The free polysaccharide content on conjugates kept at 4° C. or stored 7days at 37° C. is determined on the supernatant obtained afterincubation with α-carrier antibodies and saturated ammonium sulfate,followed by a centrifugation.

An α-PS/α-PS ELISA is used for the quantification of free polysaccharidein the supernatant. The absence of conjugate is also controlled by anα-carrier/α-PS ELISA.

Example 4 Formulation Adjuvant Compositions

Protein, either individually or together, from the above examples maybeformulated with the staphylococcal polysaccharide combination and asadjuvant, the formulation may comprise a mixture of 3 de —O-acylatedmonophosphoryl lipid A (3D-MPL) and aluminium hydroxide, or of 3 de—O-acylated monophosphoryl lipid A (3D-MPL) and aluminium phosphate, or3D-MPL and/or QS21 optionally in an oil/water emulsion, and optionallyformulated with cholesterol, or aluminium salt alone, preferablyaluminium phosphate.

3D-MPL: is a chemically detoxified form of the lipopolysaccharide (LPS)of the Gram-negative bacteria Salmonella minnesota.

Experiments performed at GSK Biologicals have shown that 3D-MPL combinedwith various vehicles strongly enhances both the humoral and a TH1 typeof cellular immunity.

QS21: is one saponin purified from a crude extract of the bark of theQuillaja Saponaria Molina tree, which has a strong adjuvant activity: itactivates both antigen-specific lymphoproliferation and CTLs to severalantigens.

Vaccine containing an antigen of the invention containing 3D-MPL andalum may be prepared in analogous manner to that described in WO93/19780or 92/16231.

Experiments performed at GSK Biologicals have demonstrated a clearsynergistic effect of combinations of 3D-MPL and QS21 in the inductionof both humoral and TH1 type cellular immune responses. Vaccinescontaining an antigen such antigens are described in U.S. Pat. No.5,750,110.

The oil/water emulsion is composed of 2 oils (a tocopherol andsqualene), and of PBS containing Tween 80 as emulsifier. The emulsioncomprised 5% squalene 5% tocopherol 0.4% Tween 80 and had an averageparticle size of 180 nm and is known as SB62 (see WO 95/17210).

Experiments performed at GSK Biologicals have proven that the adjunctionof this 0/W emulsion to MPL/QS21 further increases their immunostimulantproperties.

Preparation of Emulsion SB62 (2 Fold Concentrate)

Tween 80 is dissolved in phosphate buffered saline (PBS) to give a 2%solution in the PBS. To provide 100 ml two fold concentrate emulsion 5 gof DL alpha tocopherol and 5 ml of squalene are vortexed to mixthoroughly. 90 ml of PBS/Tween solution is added and mixed thoroughly.The resulting emulsion is then passed through a syringe and finallymicrofluidised by using an M110S microfluidics machine. The resultingoil droplets have a size of approximately 180 nm.

Example 5 Animal Experiments

Female CD-1 mice, 8 to 10 weeks old, are obtained from Charles RiverLaboratories, Kingston, Mass. For lethality studies, five groups of 9 to11 CD-1 mice are challenged intraperitoneally (i.p.) with serialdilutions of S. aureus grown on CSA plates. The inocular sizes rangefrom ˜10¹⁰ to 10⁸ CFU/mouse. Mortality is assessed on a daily basis for3 days. The 50% lethal doses (LD₅₀s) is estimated by using a probitmodel of the dose-response relationship. The null hypothesis of commonLD₅₀s was tested by the likelihood ratio test. Sublethal bacteremia isinitiated by challenging groups of 8 to 20 mice by the intravenous(i.v.) route with ˜2×10⁶ CFU/mouse or by the i.p. route with ˜2×10⁷CFU/mouse. After inoculation separate groups of animals are bled fromthe tail at specified times, and the bacteremia levels are estimated byquantitative plate counts performed in duplicate on tryptic soy agarplates with 5% sheep blood (Becton Dickinson Microbiology Systems).Statistical significance is determined with the Welch modification ofthe unpaired Stutent's t test.

Example 6 General Methodology of Determining Antibody Responses inVarious Mammals

The sera were tested for IgG antibodies to the staphylococcalpolysaccharides by an ELISA. Briefly, purified capsular polysaccharidesfrom ATCC (Rockville, Md., 20852) are coated at 25 μg/ml in phosphatebuffered saline (PBS) on high binding microtitre plates (Nunc Maxisorp)overnight at 4 C. The plates are blocked with 10% fetal calf serum(FCS), 1 hour at 37 C. Serum samples are pre-incubated with the 20 μg/mlcell-wall polysaccharide (Statens Serum Institute, Copenhagen) and 10%FCS at room temperature for 30 minutes to neutralize antibodies to thisantigen. The samples are then diluted two-fold on the microplate in 10%FCS in PBS, and equilibrated at room temperature for 1 hour withagitation. After washing, the microplates are equilibrated withperoxidase labelled anti-human IgG Fc monoclonal antibody (HP6043-HRP,Stratech Scientific Ltd) diluted 1:4000 in 10% FCS in PBS for 1 hour atroom temperature with agitation. The ELISA is performed to measure ratIgG using Jackson ImmunoLaboratories Inc. peroxidase-conjugatedAffiniPure Goat anti-Rat IgG (H+L) (code 112-035-003) at 1:5000. Thetitration curves are referenced to standard sera for each serotype usinglogistic log comparison by SoftMax Pro. The polysaccharideconcentrations used to coat the ELISA plate are 10-20 μg/ml. The coloris developed using 4 mg OPD (Sigma) per 10 ml pH 4.5 0.1M citrate bufferwith 14 μl H2O2 for 15 minutes in the dark at room temperature. Thereaction is stopped with 50 μl, HCl, and the optical density is read at490 nm relative to 650 nm. IgG concentrations are determined byreference of titration points to the calibration curve modeled using a4-parameter logistic log equation calculated by SoftMax Pro software.

The ELISA to measure the murine and rat IgG to the staphylococcalpolysaccharides is similar with the following exceptions. JacksonImmunoLaboratories Inc. peroxidase-conjugated affiniPure Goat Anti-mouseIgG (H+L) and AffiniPure Goat Anti-rat IgG (H+L) were employed to detectbound IgG.

HP6043-HRP reacts equally with human and Rhesus purified IgG, and sothis reagent is used for Rhesus antiserum.

The protein ELISA is performed similarly to the polysaccharide ELISAwith the following modifications. The protein is coated overnight at 2.0μg/ml in PBS. The serum samples are diluted in PBS containing 10% foetalcalf serum and 0.1% polyvinyl alcohol. Bound human antibody is detectedusing Sigma Peroxidase-conjugated goat affinity purified antibody toHuman IgG Fc (reference A-2290).

Example 7 Opsonophagocytosis Assay

The in vitro opsonophagocytosic killing of S. aureus by humanpolymorphonuclear leykocytes (PMNs) is performed as described in Xu etal 1992 Infect. Immun. 60; 1358. Human PMNs are prepared fromheparinized blood by sedimentation in 3% dextran T-250. The opsonicreaction mixture (1 ml) contains ˜10⁶ PMNs in RPMI 1640 mediumsupplemented with 10% heat-inactivated fetal calf serum, ˜10⁸ CFU ofS-aureus, and 0.1 ml of the test serum or IgG preparation.Hyperimmunized rabbit serum is used as a positive control, and 0.1 ml ofnonimmune rabbit serum was used as a complete source for the IgGsamples. The reaction mixtures are incubated at 37° C., and bacterialsamples are transferred at 0, 60, and 120 min into water andsubsequently diluted, spread on tryptic soy agar plates, and incubatedat 37° C. for bacterial count after overnight incubation.

Example 8 Immunogenicity of Staphylococcal Proteins in Mice and Rabbits

Animals were immunized with purified staphylococcal proteins in order togenerate hyper-immune sera. Mice were immunized three times (days 0, 14and 28) with 10 μg of each proteins adjuvanted in Specol. Rabbits wereimmunized three times (days 0, 21 and 42) with 20 μg of each proteinsadjuvanted in Specol. Immune sera were collected and evaluated inanti-protein and anti-killed whole cells ELISA.

Anti-Protein ELISA:

The purified protein was coated at 1 μg/ml in phosphate buffered saline(PBS) on high binding microtitre plates (Nunc Maxisorp) overnight at 4°C. The plates were blocked with PBS-BSA 1%, for 30 min at RT withagitation. The test samples were then diluted 1/1000 and incubated atroom temperature for 1 hour with agitation. After washing, bound murineor rabbit antibody was detected using Jackson ImmunoLaboratories Inc.peroxidase-conjugated affiniPure Goat Anti-Mouse IgG (H+L) (ref:115-035-003) or AffiniPure Goat Anti-Rabbit IgG (H+L) (ref: 11-035-003)diluted 1:5000 in PBS-tween 0.05%. The detection antibodies wereincubated for 30 min. at room temperature with agitation. The color wasdeveloped using 4 mg OPD (Sigma)+5 μl H2O2 per 10 ml pH 4.5 0.1M citratebuffer for 15 minutes in the dark at room temperature. The reaction wasstopped with 50 μl HCl, and the optical density was read at 490 nmrelative to 650 nm. The O.D. for a 1/1000 dilution of Post III wascompared to the O.D. obtained with the same dilution of Pre-immune sera.

Results generated with mice and rabbit sera are presented in FIG. 5. Agood seroconversion against each antigen was observed. Evaluation ofsera directed against SBI was impaired due to the Ig binding activity ofthis protein.

Anti-Killed Whole Cells ELISA:

Killed whole cells (heat or formaldehyde inactivated) from S. aureustype 5 and 8 or S. epidermidis strain Hay were coated at 20 μg/ml inphosphate buffered saline (PBS) on high binding microtitre plates (NuncMaxisorp) overnight at 4° C. with evaporation. The plates were blockedwith PBS-BSA 1% 30 min at room temperature with agitation. Protein A wasneutralised by addition of 10 μg/ml of Affinity Purified Chickednanti-ProteinA (ICL ref: CPA-65A-2) diluted in PBS-tween 0.05% followedby incubation for 1 hour at room temperature. The test samples were thendiluted two-fold on the microplate in PBS-0.05% from a starting dilutionat 1/10 and incubated 1 hour at room temperature with agitation. Afterwashing, bound murine or rabbit antibody was detected using JacksonImmunoLaboratories Inc. peroxidase-conjugated affiniPure Goat Anti-MouseIgG (H+L) (ref: 115-035-003) or AffiniPure Goat Anti-Rabbit IgG (H+L)(ref: 11-035-003) diluted 1:5000 in PBS-tween 0.05%. This detectionantibodies were incubated for 30 min. at room temperature withagitation. The color was developed using 4 mg OPD (Sigma)+5 μl H₂O2 per10 ml pH 4.5 0.1M citrate buffer for 15 minutes in the dark, at roomtemperature. The reaction was stopped with 50 μl HCl, and the opticaldensity was read at 490 nm relative to 650 nm.

It should be noted that expression levels of proteins in staphylococciwill vary depending on culture conditions. Therefore a negative resultmay reflect the choice of incorrect culture conditions rather than alack of immunogenicity.

The results using mice sera are shown in Table 5 and some of the graphsare shown in FIG. 6. A weak recognition of S. aureus strain 5 isobserved with sera directed against SdrC, FnbpA, Ebh, Sbi and IsaA.Recognition of S. aureus strain 8 is only observed with the serumdirected against Sbi. Weak recognition of S. epidermidis Hay is observedwith sera directed against Atl amidase, MRP, IsdA, IsaA, Ebh, Aaa andSbi.

A selection of results generated using rabbit sera are shown in FIG. 7and summarized in Table 6. Very good recognition of the three strainswas observed with IsaA and IsdB. A weak recognition of the three stainswas observed with HarA although animals only received one injectionrather than the three injections used for the other proteins.

TABLE 5 Protein name React on SA5 React on SA8 React on SE Hay IsaA (+)(+) (+) ClfA − (+) (+) Atl amidase − − ++ SdrG − − − Glucosamidase − − −IsdA − − ++ Alpha toxin − − − SrdC ++ (+) − Ebh + − + AaA − − ++ MRP − −++ Sbi ++ ++ +++ FnbpA + + (+)

TABLE 6 Protein name React on SA5 React on SA8 React on SE Hay IsaA ++++++ +++ ClfA + ++ ++ Atl amidase − ++ + IsdB +++ +++ +++ SdrG + + +Glucosamidase − − − HarA (1 inject.) + + + IsdA − − − Alpha toxin − − +SrdC − − − Ebh − + − AaA − − − MRP − − ++ Sbi − +++ − FnbpA − ++ ++

Example 8 Efficacy of Combinations of Staphylococcal Proteins in a NasalColonization Model

Fifteen groups of three cotton rats were inoculated with combinations ofeight staphylococcal antigens and five cotton rats which acted ascontrols were treated with no antigen. These sixteen groups are asfollows:

Group 1—Atl-glucosamine, Atl-amidase, AAA, alpha toxin, SdrC, SdrG, Ebh,Sbi

Group 2—Atl-glucosamine, Atl-amidase, IsdA, IsdB, ClfA, SdrC, Ebh, FnbpA

Group 3—Atl-glucosamine, Atl-amidase, HarA, IsdA, MRP, IsdB, AAA, alphatoxin

Group 4—Atl-glucosamine, HarA, IsdA, AAA, ClfA, IsaA, Ebh, Sbi

Group 5—HarA, MRP, AAA, alpha toxin, ClfA, SdrC, Ebh, FnbpAGroup 6—IsdA, IsdB, AAA, alpha toxin, ClfA, SdrG, Sbi, FnbpA

Group 7—Atl-aminidase, IsdA, MRP, AAA, IsaA, SdrG, Ebh, FnbpA Group8—Control

Group 9—Atl-glucosamine, IsdA, MRP, alpha toxin, IsaA, SdrC, Sbi, FnbpA

Group 10—Atl-glucosamine, MRP, IsdB, AAA, ClfA, IsaA, SdrC, SdrG

Group 11—Atl-amindase, MRP, IsdB, alpha toxin, ClfA, IsaA, Ebh, SbiGroup 12—Atl-glucosamine, HarA, IsdB, alpha toxin, IsaA, SdrG, Ebh,FnbpA

Group 13—Atl-amidase, HarA, IsdB, AAA, IsaA, SdrC, Sbi, FnbpA Group14—Atl-glucosamine, Atl-amidase, HarA, MRP, ClfA, SdrG, Sbi, FnbpA

Group 15—Atl-amidase, HarA, IsdA, alpha toxin, ClfA, IsaA, SdfC, SdrG

Group 16—HarA, IsdA, MRP, IsdB, SdrC, SdrG, Ebh, Sbi

Each mix of antigens contained 3 μg of each antigen mixed with anadjuvant made of liposomes containing MPL and QS21. The cotton rats wereinoculated three times on days 1, 14 and 28 of the experiment. Two weeksafter inoculation, the efficacy of the immunisations were assessed usinga nasal colonisation assay as described in Kokai-Kun et al (2003)Antimicrob. Agents. Chemother. 47; 1589-1597.

Classical multiple linear regression analysis was carried out on thedata using “Design Expert 6” software. The presence of an antigen wascoded as +1 and the absence of an antigen by −1. Using the equation ofthe model it was possible to determine which antigens were the keyantigens which produced a large decrease in the number of colonies pernose.

Results

The results of the nasal colonisation assay are shown in Table 7. Thecontrol group had a mean logCFU/nose of 3.51335 and a decrease in nasalcolonisation could be see for all the groups of cotton rats inoculatedwith staphylococcal proteins. Groups 4, 9 and 13 showed the greatestdecrease in nasal colonisation with a decrease of over 2 logs inCFU/nose. Groups 12 and 16 also gave good results, showing a decease ofabout 2 logs in CFU/nose.

TABLE 7 Group Mean observed LogCFU/nose Predicted LogCFU/nose 1 1.775272.03560 2 2.90435 2.52684 3 1.96556 2.23033 4 1.27748 1.21872 5 1.673041.93128 6 2.79745 2.98193 7 2.21481 2.30705 8 3.51355 3.47317 9 1.224801.44080 10 2.03085 1.93204 11 2.02522 1.81581 12 1.53402 1.70996 131.36063 1.49100 14 2.31201 1.73909 15 2.22979 1.98223 16 1.58109 1.44004

The contribution of specific antigens within the antigen mix wascalculated using multiple regression analysis of the nasal colonisationdata. The final mdel contains the seven best antigens. Results for theseantigens are shown in Table 8. Within the context of the protein mix,the inclusion of HarA gave the greatest decrease in nasal colonisation,followed by IsaA, Sbi, SdrC, autolysin-glucosamine, MRP and Ebh.

TABLE 8 Effects in difference of logCFU/nose and ratio of CFU/nose forthe seven best antigens in the model and corresponding p-values. EffectReduction Cumulative Cumulative antigen prob > F estimate ratio effectratio HarA 0.033 −0.596 3.9 −0.596 3.9 IsaA 0.046 −0.558 3.6 −1.154 14.3Sbi 0.077 −0.491 3.1 −1.645 44.2 SdrC 0.22 −0.337 2.2 −1.982 96.0Atl-glucos 0.238 −0.324 2.1 −2.306 202.2 MRP 0.239 −0.323 2.1 −2.629425.3 Ebh 0.297 −0.286 1.9 −2.914 821.0

1. An immunogenic composition comprising staphylococcal PNAG and Type 5and/or 8 capular polysaccharide or oligosaccharide from S. aureus. 2.The immunogenic composition of claim 1 further comprising Type I, and/orType II and/or Type III capsular polysaccharide or oligosaccharide fromS. epidermidis.
 3. The immunogenic composition of claim 1 wherein thePNAG is derived from a staphylococcal bacterium.
 4. The immunogeniccomposition of claim 1 further comprising a staphylococcal protein orfragment thereof.
 5. The immunogenic composition of claim 4 wherein thestaphylococcal protein or fragment thereof is an extracellular componentbinding protein selected from, the group consisting of laminin receptor,SitC/MntC/saliva binding protein, EbhA, EbhB, Elastin binding protein(EbpS), EFB (FIB), SBI, autolysin; ClfA, SdrC, SdrG, SdrH, Lipase GehD,SasA, FnbA, Cna; ClfB, FbpA, Npase, IsaAIPisA, SsaA, EPB, SSP-1, SSP-2,HBP, Vitronectin binding protein, fibrinogen binding protein, coagulase,Fig and MAP.
 6. The immunogenic composition of claim 4 wherein thestaphylococcal protein or fragment thereof is a transporter proteinselected from the group consisting of Immunodominant ABC transporter,IsdA, 15 dB, Mg2+ transporter, SitC and Ni ABC transporter.
 7. Theimmunogenic composition of claim 4 wherein the staphylococcal protein orfragment thereof is a toxin or regulator of virulence selected from thegroup consisting of alpha toxin (Hla), alpha toxin H35R mutant, RNA IIIactivating protein (RAP).
 8. The immunogenic composition of claim 4comprising 2 or more staphylococcal proteins selected from at least 2different groups selected from; a) at least one staphylococcalextracellular component binding protein or fragment thereof selectedfrom the group consisting of laminin receptor, SitC/MntC/saliva bindingprotein, EbhA, EbhB, Elastin binding protein (EbpS), EFB (FIB), SBI,autolysin, ClfA, SdrC, SdrG, SdrH, Lipase GehD, SasA, FnbA, FnbB, Cna,ClfB, FbpA, Npase, IsaA/PisA, SsaA, EPB, SSP-1, SSP-2, Vitronectinbinding protein, fibrinogen binding protein, coagulase, Fig and MAP; b)at least one staphylococcal transporter protein or fragment thereofselected from the group consisting of Immunodominant ABC transporter,IsdA, IsdB, Mg2+ transporter, SitC and Ni ABC transporter; c) at leastone staphylococcal regulator of virulence, toxin or fragment thereofselected from the group consisting of alpha toxin (Hla), alpha toxinH35R mutant, RNA III activating protein' (RAP).
 9. The immunogeniccomposition of claim 1 wherein a staphylococcal polysaccharide isconjugated to a protein carrier.
 10. The immunogenic composition ofclaim 1 wherein the PNAG is conjugated to a protein carrier.
 11. Theimmunogenic composition of claim 9 wherein the protein carrier comprisesa staphylococcal protein or fragment thereof selected from the groupconsisting of laminin receptor, SitC/MntC/saliva binding protein, EbhA,EbhB, Elastin binding protein (EbpS), EFB (FIB), SBI, autolysin, ClfA,SdrC, SdrG, SdrH, Lipase GehD, SasA, FnbA, FnbB, Cna, ClfB, FbpA, Npase,IsaA/PisA, SsaA, EPB, SSP-1, SSP-2, HBP, Vitronectin binding protein,fibrinogen binding protein, coagulase, Fig, MAP, Immunodominant ABCtransporter, IsdA, IsdB, Mg2+ transporter, SitC and Ni ABC transporter,alpha toxin (Hla), alpha toxin H35R mutant and RNA III activatingprotein (RAP).
 12. The immunogenic composition of claim 9 wherein theprotein carrier is selected from the group consisting of tetanus toxoid,diphtheria toxoid, CRMI97, Haemophilus influenzae protein D, Pseudomonasaeruginosa exoprotein A, pneumococcal pneumolysin and alpha toxoid. 13.The immunogenic composition of claim 1 wherein an effective immuneresponse is generated against both S. aureus and S. epidermidis.
 14. Avaccine comprising the immunogenic composition of claim 1 and apharmaceutically acceptable excipient.
 15. A method of making a vaccinecomprising the steps of mixing antigens to make the immunogeniccomposition of claim 1 and adding a pharmaceutically acceptableexcipient.
 16. A method of preventing or treating staphylococcalinfection comprising the step of administering the vaccine of claim 14to a patient in need thereof.
 17. A use of the immunogenic compositionof claim 1 in the manufacture of a vaccine for treatment or preventionof staphylococcal infection.